Engineered artificial antigen presenting cells for tumor infiltrating lymphocyte expansion

ABSTRACT

In some embodiments, compositions and methods relating to isolated artificial antigen presenting cells (aAPCs) are disclosed, including aAPCs comprising a myeloid cell transduced with one or more viral vectors, such as a MOLM-14 or a EM-3 myeloid cell, wherein the myeloid cell endogenously expresses HLA-AB/C, ICOS-L, and CD58, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL and/or OX40L and transduce the myeloid cell to express CD86 and 4-1BBL and/or OX40L proteins. In some embodiments, methods of expanding tumor infiltrating lymphocytes (TILs) with aAPCs and methods of treating cancers using TILs after expansion with aAPCs are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No. PCT/US17/59271, filed Oct. 31, 2017, which claims the benefit of priority to U.S. Provisional Application No. 62/481,831, filed Apr. 5, 2017, U.S. Provisional Application No. 62/475,053, filed Mar. 22, 2017, U.S. Provisional Application No. 62/438,600, filed Dec. 23, 2016, and U.S. Provisional Application No. 62/415,274, filed Oct. 31, 2016, the entireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

Engineered artificial antigen presenting cells (aAPCs) for expansion of tumor infiltrating lymphocytes are disclosed.

BACKGROUND OF THE INVENTION

Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. A large number of TILs are required for successful immunotherapy, and a robust and reliable process is needed for commercialization. This has been a challenge to achieve because of technical, logistical, and regulatory issues with cell expansion. IL-2-based TIL expansion followed by a “rapid expansion process” (REP) has become a preferred method for TIL expansion because of its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. However, although REP can result in a 1,000-fold expansion of TILs over a 14-day period, it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. Despite their high performance, PBMCs have multiple drawbacks, including the large numbers of allogeneic PBMCs required, the need to obtain PBMCs by leukapheresis from multiple healthy donors, the resulting interdonor variability in PBMC viability after cryopreservation and variable TIL expansion results, the risk of undetected viral pathogens causing downstream patient infections, and the extensive and costly laboratory testing of each individual donor cell product to confirm sterility and quality (including viral contaminant testing) and to test expansion properties.

Unfortunately, aAPCs developed for use in the expansion of TILs have suffered from poor performance when compared to PBMCs, including alterations of the phenotypic properties of the input TILs, as well as poor expansion performance and/or high variability in expansion results. Because of the large number of potential cells that might be adapted for use as aAPCs and the unpredictability of identifying suitable candidates, the focus of aAPC development for polyclonal TILs to date has been solely on the well-established K562 cell line. Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. For example, K562 cells modified to express 4-1BBL (CD137L) were tested in pre-REP culture (but not in REP culture) to determine enhancement of TIL expansion from tumor digest, but PBMCs were still required to be used in conjunction with K562 cells to obtain TIL expansion. Friedman, et al., J. Immunother. 2011, 34, 651-661. Other engineered K562 cells modified to express CD64, CD86, and 4-1BBL were tested and achieved TIL expansion that was at best comparable to PBMCs, and most likely less than PBMCs, and also suffered from skewing of the polyclonal TIL phenotype to a less favorable CD8⁺/CD4⁺ T cell ratio. Ye, et al., J. Translat. Med. 2011, 9, 131. Recently, K562 cells modified to express CD86, 4-1BBL (CD137L), high affinity Fc receptor (CD64) and membrane-bound IL-15 have also been shown to propagate TIL (post-REP) at equivalent numbers compared to PBMC feeders, but with the additional complexity of membrane-bound IL-15. Forget, et al., J. Immunother. 2014, 37, 448-60. Other systems developed have lacked critical costimulatory molecules, have led to unfavorable T cell phenotypic skewing, or have required additional interleukins (such as IL-21). Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. Overall, K562 modified aAPCs have not been shown to provide for consistent expansion of TILs with acceptable variability while also performing better than PBMCs in other measures including overall expansion cell counts. Alternative aAPCs besides K562 cells have been successful in other cell expansion methods, but have not achieved the same performance as PBMCs with the unique polyclonal subset of cells that make up TILs. Maus, et al., Nat. Biotechnol. 2002, 20, 143-148; Suhoski, et al., Mol. Ther. 2007, 15, 981-988.

The MOLM-14 human leukemia cell line was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-A/B/C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86. MOLM-14 cells and the closely-related MOLM-13 cells have not been previously reported as useful aAPCs for the expansion of cells for tumor immunotherapy applications.

The EM-3 human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CML. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. EM-3 cells and the closely-related EM-2 cell line have not been previously reported as useful aAPCs for the expansion of cells for tumor immunotherapy applications. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33.

The present invention provides the unexpected finding that engineered myeloid lineage cells, including MOLM-13, MOLM-14, EM-3, and EM-2 cells, transduced with additional costimulatory molecules, including CD86 (B7-2), 4-1BBL (CD137L), and OX40L (CD134L), provide for superior and highly efficient expansions of TILs in large numbers with minimal variability, reduced cost, and no reliance on human blood samples as a source of PBMCs, with the benefit of using an aAPC which can be produced efficiently from a master cell bank. CD86 and 4-1BBL are costimulatory molecules that provide costimulatory signals for T cell activation. The MOLM-14, MOLM-13, EM-3, and/or EM-2 cells transduced with additional costimulatory molecules are useful, for example, in the expansion of TILs for use in cancer immunotherapy and other therapies.

SUMMARY OF THE INVENTION

In an embodiment, the invention provides an artificial antigen presenting cell (aAPC) comprising a myeloid cell transduced with one or more vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein.

In an embodiment, each of the CD86 protein and the 4-1BBL protein are human proteins.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC can stimulate and expand a tumor infiltrating lymphocyte (TIL) contacted with the aAPC.

It will be apparent that in certain embodiments of the invention, the nucleic acid molecule encoding CD86 may be comprised in a different viral vector to the nucleic acid molecule encoding 4-1BBL or the same viral vector.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 IU/mL and OKT-3 antibody at a concentration of about 30 ng/mL.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC can stimulate and expand a T cell contacted with the aAPC.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell endogenously expresses HLA-A/B/C, ICOS-L, and CD58.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is essentially devoid of membrane-bound IL-15.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a MOLM-14 cell.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a MOLM-13 cell.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a EM-3 cell.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a EM-2 cell.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the CD86 protein comprises an amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof, and the 4-1BBL protein comprises SEQ ID NO:9, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof.

In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the nucleic acid molecule encoding CD86 comprises a nucleic acid sequence as set forth in SEQ ID NO:16 and the nucleic acid molecule encoding 4-1BBL comprises a nucleic acid sequence as set forth in SEQ ID NO:19.

In an embodiment, the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising the step of contacting a population of TILs with an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and wherein the population of TILs is expanded. In an embodiment, the method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the cell culture medium         further comprises IL-2 at an initial concentration of about 3000         IU/mL and OKT-3 antibody at an initial concentration of about 30         ng/mL.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the population of APCs         expands the population of TILs by at least 50-fold over a period         of 7 days in a cell culture medium.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the myeloid cell         endogenously expresses HLA-A/B/C, ICOS-L, and CD58.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the myeloid cell is a         MOLM-14 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the myeloid cell is a         MOLM-13 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the myeloid cell is a         EM-3 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the myeloid cell is a         EM-2 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivo method.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid molecule encoding CD86 and a nucleic acid molecule         encoding 4-1BBL, and wherein the myeloid cell expresses a CD86         protein and a 4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the CD86 protein         comprises an amino acid sequence as set forth in SEQ ID NO:8, or         comprises an amino acid sequence comprising one or more         conservative amino acid substitutions thereof, and the 4-1BBL         protein comprises an amino acid sequence as set forth in SEQ ID         NO:9, or comprises an amino acid sequence comprising one or         conservative amino acid substitutions thereof.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the nucleic acid         encoding CD86 comprises a nucleic acid sequence as set forth in         SEQ ID NO:16 and the nucleic acid encoding 4-1BBL comprises a         nucleic acid sequence as set forth in SEQ ID NO:19.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the expansion is         performed using a gas permeable container.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the ratio of the         population of TILs to the population of aAPCs is between 1 to         200 and 1 to 400.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium, wherein the ratio of the         population of TILs to the population of aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a myeloid artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co-stimulatory molecules on the TILs, wherein binding of the co-stimulatory molecules with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least two co-stimulatory ligands comprise CD86 and 4-1BBL. In an embodiment, the foregoing method is an in vitro or ex vivo method.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced         to express a CD86 protein and a 4-1BBL protein.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating cancer, wherein the TILs are a second population of TILs and are obtainable from a method comprising the steps of:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the TILs are/have been         obtained from a tumor resected from a patient, and wherein the         second population of TILs is at least 50-fold greater in number         than the first population of TILs after 7 days from the start of         the rapid expansion; and     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced         to express a CD86 protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, wherein the myeloid         aAPCs comprise MOLM-14 cells transduced with one or more viral         vectors, and wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the MOLM-14 cells express a CD86 protein and a         4-1BBL protein.

In an embodiment, the invention provides a population of tumor infiltrating cells (TILs) for use in treating a cancer, wherein the population of TILs is a second population of TILs and is obtainable by a process comprising:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the first population of TILs         are/have been obtained from a tumor resected from a patient,         wherein the second population of TILs is at least 50-fold         greater in number than the first population of TILs after 7 days         from the start of the rapid expansion;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, wherein the myeloid         aAPCs comprise MOLM-14 cells transduced with one or more viral         vectors, and wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the MOLM-14 cells express a CD86 protein and a         4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, wherein the myeloid         aAPCs comprise EM-3 cells transduced with one or more viral         vectors, and wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the EM-3 cells express a CD86 protein and a 4-1BBL         protein.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the first population of TILs         are/have been obtained from a tumor resected from a patient, and         wherein the second population of TILs is at least 50-fold         greater in number than the first population of TILs after 7 days         from the start of the rapid expansion; and     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, wherein the myeloid         aAPCs comprise EM-3 cells transduced with one or more viral         vectors, and wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the EM-3 cells express a CD86 protein and a 4-1BBL         protein.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, and wherein the         rapid expansion is performed over a period not greater than 14         days.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a second population and is obtainable by a method comprising the steps of:

-   -   (a) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion,         wherein the myeloid aAPCs endogenously express HLA-A/B/C, ICOS-L         and CD58, wherein the myeloid aAPCs are transduced to express a         CD86 protein and a 4-1BBL protein, and wherein the rapid         expansion is performed over a period not greater than 14 days.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, and wherein the         cell culture medium further comprises IL-2 at an initial         concentration of about 3000 IU/mL and OKT-3 antibody at an         initial concentration of about 30 ng/mL.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the first population of TILs         are/have been obtained from a tumor resected from a patient, and         wherein the second population of TILs is at least 50-fold         greater in number than the first population of TILs after 7 days         from the start of the rapid expansion; and wherein the myeloid         aAPCs endogenously express HLA-A/B/C, ICOS-L, and CD58, wherein         the myeloid aAPCs are transduced to express a CD86 protein and a         4-1BBL protein, and wherein the cell culture medium further         comprises IL-2 at an initial concentration of about 3000 IU/mL         and OKT-3 antibody at an initial concentration of about 30         ng/mL.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, and wherein the         expansion is performed using a gas permeable container.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the first population of TILs         are/have been obtained from a tumor resected from a patient, and         wherein the second population of TILs is at least 50-fold         greater in number than the first population of TILs after 7 days         from the start of the rapid expansion; and wherein the myeloid         aAPCs endogenously express HLA-A/B/C, ICOS-L, and CD58, wherein         the myeloid aAPCs are transduced to express a CD86 protein and a         4-1BBL protein, and wherein the expansion is performed using a         gas permeable container.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, and wherein the         ratio of the second population of TILs to the population of         aAPCs is between 1 to 200 and 1 to 400.

In an embodiment, the invention provides a population of tumor infiltrating cells (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising the steps of:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the first population of TILs         is/has been obtained from a tumor resected from a patient, and         wherein the second population of TILs is at least 50-fold         greater in number than the first population of TILs after 7 days         from the start of the rapid expansion; and     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, and wherein the         ratio of the second population of TILs to the population of         aAPCs is between 1 to 200 and 1 to 400. In certain embodiments,         the ratio of the second population of TILs to the population of         aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, and wherein the         ratio of the second population of TILs to the population of         aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing a rapid expansion of the first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain a         second population of TILs, wherein the second population of TILs         is at least 50-fold greater in number than the first population         of TILs after 7 days from the start of the rapid expansion; and     -   (c) administering a therapeutically effective portion of the         second population of TILs to a patient with the cancer;     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, wherein the cancer         is selected from the group consisting of melanoma, ovarian         cancer, cervical cancer, non-small-cell lung cancer (NSCLC),         lung cancer, bladder cancer, breast cancer, cancer caused by         human papilloma virus, head and neck cancer, renal cancer, and         renal cell carcinoma.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a method comprising the steps of:

-   -   (a) performing a rapid expansion of a first population of TILs         using a population of myeloid artificial antigen presenting         cells (myeloid aAPCs) in a cell culture medium to obtain the         second population of TILs, wherein the first population of TILs         is/has been obtained from a tumor resected from a patient, and         wherein the second population of TILs is at least 50-fold         greater in number than the first population of TILs after 7 days         from the start of the rapid expansion; and     -   wherein the myeloid aAPCs endogenously expresses HLA-A/B/C,         ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to         express a CD86 protein and a 4-1BBL protein, wherein the cancer         is selected from the group consisting of melanoma, ovarian         cancer, cervical cancer, non-small-cell lung cancer (NSCLC),         lung cancer, bladder cancer, breast cancer, cancer caused by         human papilloma virus, head and neck cancer, renal cancer, and         renal cell carcinoma.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the myeloid aAPCs comprise MOLM-14 cells transduced with         one or more viral vectors, wherein the one or more viral vectors         comprise a nucleic acid encoding CD86 and a nucleic acid         encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86         protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the myeloid aAPCs comprise EM-3 cells transduced with         one or more viral vectors, wherein the one or more viral vectors         comprise a nucleic acid encoding CD86 and a nucleic acid         encoding 4-1BBL, and wherein the EM-3 cells express a CD86         protein and a 4-1BBL protein.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and obtainable by a method comprising the steps of:

-   -   (a) performing an initial expansion of a first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the first population of TILs is/has         been obtained from a tumor resected from a patient, and wherein         the second population of TILs is at least 5-fold greater in         number than the first population of TILs, and wherein the first         cell culture medium comprises IL-2;     -   (b) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain the         third population of TILs, wherein the third population of TILs         is at least 50-fold greater in number than the second population         of TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3.

In an embodiment, the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the myeloid cells comprise MOLM-13 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid cells comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid cells comprise EM-2 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-2 cells express a CD86 protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) treating the patient with a non-myeloablative         lymphodepletion regimen, wherein the non-myeloablative         lymphodepletion regimen comprises the steps of administration of         cyclophosphamide at a dose of 60 mg/m²/day for two days followed         by administration of fludarabine at a dose of 25 mg/m²/day for         five days;     -   (e) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer; and     -   (f) treating the patient with a high-dose IL-2 regimen, wherein         the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of         aldesleukin administered as a 15-minute bolus intravenous         infusion every eight hours until tolerance;     -   wherein the myeloid aAPCs comprise MOLM-14 cells transduced with         one or more viral vectors, wherein the one or more viral vectors         comprise a nucleic acid encoding CD86 and a nucleic acid         encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86         protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) treating the patient with a non-myeloablative         lymphodepletion regimen, wherein the non-myeloablative         lymphodepletion regimen comprises the steps of administration of         cyclophosphamide at a dose of 60 mg/m²/day for two days followed         by administration of fludarabine at a dose of 25 mg/m²/day for         five days;     -   (e) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer; and     -   (f) treating the patient with a high-dose IL-2 regimen, wherein         the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of         aldesleukin administered as a 15-minute bolus intravenous         infusion every eight hours until tolerance;     -   wherein the myeloid aAPCs comprise EM-3 cells transduced with         one or more viral vectors, wherein the one or more viral vectors         comprise a nucleic acid encoding CD86 and a nucleic acid         encoding 4-1BBL, and wherein the EM-3 cells express a CD86         protein and a 4-1BBL protein.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs are a third population of TILs and obtainable by a method comprising the steps of:

-   -   (a) an initial expansion of a first population of TILs in a         first cell culture medium to obtain a second population of TILs,         wherein the first population of TILs is/has been obtained from a         tumor resected from a patient, and wherein the second population         of TILs is at least 5-fold greater in number than the first         population of TILs, and wherein the first cell culture medium         comprises IL-2; and     -   (b) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain the         third population of TILs, wherein the third population of TILs         is at least 50-fold greater in number than the second population         of TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         and further wherein the population of TILs is for administration         to a patient in combination with a non-myeloablative         lymphodepletion regimen, wherein the non-myeloablative         lymphodepletion regimen comprises cyclophosphamide which is for         administration at a dose of 60 mg/m²/day for two days followed         by fludarabine which is for administration at a dose of 25         mg/m²/day for five days and further wherein the population of         TILs is for administration in combination with a high-dose IL-2         regimen, wherein the high-dose IL-2 regimen comprises 600,000 or         720,000 IU/kg of aldesleukin for administration as a 15-minute         bolus intravenous infusion every eight hours until tolerance. In         certain embodiments, the population of TILs is for         administration prior to the high-dose IL-2 regimen and         subsequent to the non-myeloablative lymphodepletion regimen.

In certain embodiments, the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. the myeloid aAPCs comprise MOLM-13 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.

In an embodiment, the population of TILs is for use in the treating of a cancer selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         and     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein IL-2 is present at an initial concentration of about         3000 IU/mL and OKT-3 antibody is present at an initial         concentration of about 30 ng/mL in the second cell culture         medium.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the first population of TILs is/has         been obtained from a tumor resected from a patient, and wherein         the second population of TILs is at least 5-fold greater in         number than the first population of TILs, and wherein the first         cell culture medium comprises IL-2; and     -   (b) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain the         third population of TILs, wherein the third population of TILs         is at least 50-fold greater in number than the second population         of TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         wherein IL-2 is present at an initial concentration of about         3000 IU/mL and OKT-3 antibody is present at an initial         concentration of about 30 ng/mL in the second cell culture         medium.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         and     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the rapid expansion is performed over a period not         greater than 14 days.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the first population of TILs is/has         been obtained from a tumor resected from a patient, and wherein         the second population of TILs is at least 5-fold greater in         number than the first population of TILs, and wherein the first         cell culture medium comprises IL-2; and     -   (b) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain the         third population of TILs, wherein the third population of TILs         is at least 50-fold greater in number than the second population         of TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         wherein the rapid expansion is performed over a period not         greater than 14 days.

In embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         and     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the initial expansion is performed using a gas permeable         container.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         and     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the rapid expansion is performed using a gas permeable         container.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the first population of TILs is/has         been obtained from a tumor resected from a patient, and wherein         the second population of TILs is at least 5-fold greater in         number than the first population of TILs, and wherein the first         cell culture medium comprises IL-2;     -   (b) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain the         third population of TILs, wherein the third population of TILs         is at least 50-fold greater in number than the second population         of TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;         wherein the initial expansion and/or the rapid expansion is         performed using a gas-permeable container.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the ratio of the second population of TILs to the         population of aAPCs in the rapid expansion is between 1 to 80         and 1 to 400.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the ratio of the second population of TILs to the         population of aAPCs in the rapid expansion is about 1 to 300.

In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the first population of TILs is/has         been obtained from a tumor resected from a patient, and wherein         the second population of TILs is at least 5-fold greater in         number than the first population of TILs, and wherein the first         cell culture medium comprises IL-2;     -   (b) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain the         third population of TILs, wherein the third population of TILs         is at least 50-fold greater in number than the second population         of TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3,         and wherein the ratio of the second population of TILs to the         population of aAPCs in the rapid expansion is between 1 to 80         and 1 to 400.

In an embodiment, the the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is about 1 to 300.

In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected         from a patient;     -   (b) performing an initial expansion of the first population of         TILs in a first cell culture medium to obtain a second         population of TILs, wherein the second population of TILs is at         least 5-fold greater in number than the first population of         TILs, and wherein the first cell culture medium comprises IL-2;     -   (c) performing a rapid expansion of the second population of         TILs using a population of myeloid artificial antigen presenting         cells (aAPCs) in a second cell culture medium to obtain a third         population of TILs, wherein the third population of TILs is at         least 50-fold greater in number than the second population of         TILs after 7 days from the start of the rapid expansion; and         wherein the second cell culture medium comprises IL-2 and OKT-3;     -   (d) administering a therapeutically effective portion of the         third population of TILs to a patient with the cancer,     -   wherein the cancer is selected from the group consisting of         melanoma, ovarian cancer, cervical cancer, non-small-cell lung         cancer (NSCLC), lung cancer, bladder cancer, breast cancer,         cancer caused by human papilloma virus, head and neck cancer,         renal cancer, and renal cell carcinoma.

In an embodiment, the invention provides a kit for specifically inducing proliferation of a tumor infiltrating lymphocyte expressing a known co-stimulatory molecule, the kit comprising an effective amount of an aAPC, wherein said aAPC comprises a MOLM-14 cell or a EM-3 cell transduced using a lentiviral vector (LV), wherein the LV comprises a nucleic acid encoding at least one co-stimulatory ligand that specifically binds said known co-stimulatory molecule, wherein binding of the known co-stimulatory molecule with said co-stimulatory ligand stimulates and expands said T cell, the kit further comprising an applicator and an instructional material for the use of said kit.

In an embodiment, the invention provides a method for assessing the potency of tumor infiltrating lymphocytes (TILs) comprising the steps of:

-   -   (a) providing a plurality of mouse mastocytoma P815 cells         expressing the endogenous CD16 Fc receptor, wherein the P815         cells are transduced with a lentiviral vector based on enhanced         green fluorescent protein (GFP) and Firefly Luciferase;     -   (b) co-culturing the plurality of P815 cells TILs with and         without OKT-3 to assess T cell receptor (TCR) activation         (specific killing) or lymphokine activated killing (LAK,         non-specific killing), respectively;     -   (c) incubating for four hours;     -   (d) adding Luciferin and incubating for 5 minutes;     -   (e) reading bioluminescence intensity using a luminometer; and     -   (f) and calculating percent cytotoxicity and survival.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

FIG. 1 illustrates the results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells. Each TIL line (M1015T and M1016T) (1.3×10⁵ cells) was co-cultured with 46 different irradiated feeders (1.3×10⁷ cells), IL-2 (3000 IU/mL) and OKT-3 (30 ng/mL) in a T25 flask for 7 days. The fold expansion value for TILs was calculated on Day 7. The figure shows the number of fold expansions for two TIL lines in separate stimulation experiments, with 46 different feeder lots tested, and highlights the variability of expansion results using PBMC feeder cells.

FIG. 2 illustrates a vector diagram of the pLV430G human 4-1BBL vector.

FIG. 3 illustrates a diagram of the 4-1BBL PCRP (polymerase chain reaction product) portion of the pLV430G human 4-1BBL vector.

FIG. 4 illustrates a vector diagram of the pLV430G human CD86 vector.

FIG. 5 illustrates a diagram of the CD86 PCRP portion of the pLV430G human CD86 vector.

FIG. 6 illustrates a vector diagram of the pDONR221 human CD86 donor vector.

FIG. 7 illustrates a vector diagram of the pDONR221 human 4-1BBL donor vector.

FIG. 8 illustrates a vector diagram of the pLV430G empty vector.

FIG. 9 illustrates a vector diagram of the pDONR221 empty vector.

FIG. 10 illustrates a vector diagram of the psPAX2 helper plasmid for lentivirus production.

FIG. 11 illustrates a vector diagram of the pCIGO-VSV.G helper plasmid for lentivirus production.

FIG. 12 illustrates the results of flow cytometry experiments on MOLM-14 cells before lentiviral transfection (“Untransfected”) and after transfection (“Transfected”), confirming the expression of CD137 and CD86 on engineered MOLM-14 cells.

FIG. 13 illustrates the results of rapid expansion of TILs using irradiated parental unmodified MOLM-14 cells (“Parent MOLM14”), engineered MOLM-14 cells (CD86/4-1BBL, “Engineered MOLM14”), or PBMC feeders (“Feeders”) for TIL lot M1032-T2. TIL were co-cultured with PBMC feeders or parental or engineered MOLM14 cells at 1:100 ratios with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11. Each dot represents cell numbers determined on Day 0, 6, 11 and 14 respectively. A logarithmic scale is used.

FIG. 14 illustrates results as shown in FIG. 13, depicted using a linear scale.

FIG. 15 illustrates results for TIL lot M1033-T6 with other parameters as given in FIG. 13, using a logarithmic scale.

FIG. 16 illustrates results as shown in FIG. 14, depicted using a linear scale.

FIG. 17 illustrates the results of rapid expansions of TILs using engineered MOLM-14 cells expressing CD86 and 4-1BBL (“TIL+Engineered MOLM14 (CD86/41BB)+OKT3”) or irradiated PBMC feeders (“TIL+Feeders+OKT3”). TIL were co-cultured with PBMC feeders or engineered MOLM-14 cells (aMOLM14) at 1:100 ratios plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11. Each point represents cell numbers determined on Day 14.

FIG. 18 illustrates the results of experiments in which TILs (2×10⁴) were cultured with different ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”, respectively) of parental MOLM-14 (“MOLM14”) cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in wells of a 24-well G-Rex plate. A control was performed using only OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) (“PBMC−”). Each condition was cultured in triplicate. Cultures were fed with fresh media and IL-2 on Day 4 and 7. Viable cells were counted on Day 7. The bar graph represented here shows the mean plus standard deviation (SD) of viable cell numbers counted on Day 11. The p-value was calculated by the student ‘t’ test.

FIG. 19 illustrates the results of TILs cultured with different ratios (1:30, 1:100, and 1:300, denoted “30”, “100”, and “300”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Viable cells were counted on day 11 and plotted. Other conditions are as in FIG. 18.

FIG. 20 illustrates the results of TILs cultured with different ratios (1:50, 1:100, and 1:200, denoted “50”, “100”, and “200”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Cells were counted on day 14. Other conditions are as in FIG. 18.

FIG. 21 illustrates the results of TILs cultured with different ratios (1:100, 1:200, 1:400, and 1:800, denoted “100”, “200”, “400”, and “800”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Cells were counted on day 14. Other conditions are as in FIG. 18.

FIG. 22 illustrates a sunburst visualization showing fine distribution of Live, T cell receptor (TCR) α/β, CD4, CD8, CD27, CD28, and CD57 TILs expanded with PBMC feeders.

FIG. 23 illustrates a sunburst visualization showing fine distribution of Live, TCR α/β, CD4, CD8, CD27, CD28, and CD57 TILs expanded with aMOLM14 aAPCs.

FIG. 24 depicts a flow cytometry contour plot showing memory subset (CD45RA+/−, CCR7+/−) gated on Live, TCR α/β+, CD4⁺, or CD8⁺ TILs.

FIG. 25 illustrates phenotypic characterization of the T cell subset, CD4⁺ and CD8⁺ post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3⁺ cells using a SPADE tree. The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIM3, PD1, and CD137.

FIG. 26 illustrates phenotypic characterization of the T cell subset, CD4⁺ and CD8⁺ post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3⁺ cells using a SPADE tree. The color gradient is proportional to the MFI CD69, CD154, KLRG1, and TIGIT

FIG. 27 illustrates oxygen consumption rate (OCR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean±standard error of the mean (SEM) measured in triplicate.

FIG. 28 illustrates extracellular acidification rate (ECAR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean±SEM measured in triplicate.

FIG. 29 illustrates a vector diagram of the destination vector pLV4301G.

FIG. 30 illustrates a vector diagram of donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII L1R5.

FIG. 31 illustrates a vector diagram of donor vector 2, pMK hCD8a scaffold TN L5 L2.

FIG. 32 illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.

FIG. 33 illustrates a vector diagram of the destination vector pLV4301G.

FIG. 34 illustrates a vector diagram of donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5.

FIG. 35 illustrates a vector diagram of donor vector 2, pMK hCD8a scaffold TN L5 L2.

FIG. 36 illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.

FIG. 37 illustrates the results of flow cytometry experiments on EM-3 cells before lentiviral transfection (“Untransfected”) and after transfection (“Transfected”), confirming the expression of CD137 and CD86 on engineered EM-3 cells.

FIG. 38 illustrates the results of experiments wherein TILs were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.

FIG. 39 illustrates the results of experiments wherein TILs were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.

FIG. 40 illustrates the results of experiments wherein TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL), with or without OKT-3 (30 ng/mL). The bar graph shows cell numbers determined on Day 11.

FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs at different TIL:aAPC ratios.

FIG. 42 illustrates the results of TIL expansions with EM-3 aAPCs. TILs (2×10⁴) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at a 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. Viable cells were counted on Day 14. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.

FIG. 43 illustrates the results of TIL expansions with EM-3 aAPCs and MOLM-14 aAPCs. TILs (2×10⁴) were co-cultured with five different PBMC feeder lots or aMOLM14 (in triplicate) or aEM3 (also in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.

FIG. 44 illustrates a sunburst visualization to show fine distribution of Live, TCR α/β, CD4⁺, and CD8⁺ TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1054).

FIG. 45 illustrates the sunburst visualization to show fine distribution of Live, TCR α/β, CD4⁺, and CD8⁺ TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1055).

FIG. 46 illustrates the CD4⁺ and CD8⁺ SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3⁺ cells. The color gradient is proportional to the MFI of LAG-3, TIM-3, PD-1, and CD137.

FIG. 47 illustrates the CD4⁺ and CD8⁺ SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3⁺ cells. The color gradient is proportional to the MFI of CD69, CD154, KLRG1, and TIGIT.

FIG. 48 illustrates a summary of spare respiratory capacity measured by the Seahorse XF Mito stress test.

FIG. 49 illustrates a summary of glycolytic reserve measured by the Seahorse XF Mito stress test.

FIG. 50 illustrates a mitochondrial stain of live TILs expanded against PBMC or aEM3 using MitoTracker dye, which stains mitochondria in live cells and for which accumulation is dependent upon membrane potential. TILs expanded against PBMC or aEM3 were stained L/D Aqua followed by MitoTracker red dye. Data shown are MitoTracker positive (MFI) cells gated on live population.

FIG. 51 illustrates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs.

FIG. 52 illustrates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs.

FIG. 53 illustrates IFN-γ release for two batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. *p<0.05, **p<0.005, ***p<0.001, ns=not significant.

FIG. 54 illustrates IFN-γ release for three batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. *p<0.05, **p<0.005, ***p<0.001, ns=not significant.

FIG. 55 illustrates Granzyme B release for two batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. *p<0.05, **p<0.005, ***p<0.001, ns=not significant.

FIG. 56 illustrates Granzyme B release for three batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. *p<0.05, **p<0.005, ***p<0.001, ns=not significant.

FIG. 57 illustrates a TIL expansion and treatment process. aAPCs of the present invention may be used in both the pre-REP stage (top half of figure) or REP stage (bottom half of figure) and may be added when IL-2 is added to each cell culture. Step 1 refers to the addition of 4 tumor fragments into 10 G-Rex 10 flasks. At step 2, approximately 40×10⁶ TILs or greater are obtained. At step 3, a split occurs into 36 G-Rex 100 flasks for REP. TILs are harvested by centrifugation at step 4. Fresh TIL product is obtained at step 5 after a total process time of approximate 43 days, at which point TILs may be infused into a patient.

FIG. 58 illustrates a treatment protocol for use with TILs expanded with aAPCs. Surgery (and tumor resection) occurs at the start, and lymphodepletion chemo refers to non-myeloablative lymphodepletion with chemotherapy as described elsewhere herein.

FIG. 59 illustrates Bioluminescent Redirected Lysis Assay (BRLA) results, showing percentage cytotoxicity of TIL batch M1033T-1 when co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios.

FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) data showing amount of IFN-γ released against different ratios of effector to target cells.

FIG. 61 illustrates LAMP1 (%) expressed by TIL batch M1033T-1 when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4 hr and 24 hr co-culture.

FIG. 62 illustrates BRLA results for TIL batch M1030. Cytotoxicity (measured as LU₅₀/1×10⁶ TIL) by BRLA is 26±16.

FIG. 63 illustrates standard chromium release assay for TIL batch M1030. Cytotoxicity (measured as LU₅₀/1×10⁶ TIL) by the chromium release assay is 22.

FIG. 64 illustrates BRLA results for TIL batch M1053, showing the lytic units of the TILs by BRLA as 70±17.

FIG. 65 illustrates standard chromium release assay results for TIL batch M1053, also showing lytic unit of the TILs by chromium assay as 14±5. Comparison of this result with FIG. 64 shows the comparable performance of the BRLA and chromium release assay.

FIG. 66 illustrates the linear relationship between IFN-γ release and cytotoxic potential of TILs.

FIG. 67 illustrates ELISpot results for IFN-γ.

FIG. 68 illustrates enzymatic IFN-γ release for TIL batch M1053.

FIG. 69 illustrates enzymatic IFN-γ release for TIL batch M1030.

FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053T and M1030T. This data confirms the potency of the TILs shown by the BRLA.

FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053.

FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030.

FIG. 73 illustrates ELISpot data showing TNF-α release by M1053T and M1030T. This data confirms the potency of the TILs shown by the BRLA.

FIG. 74 illustrates enzymatic TNF-α release for TIL batch M1053.

FIG. 75 illustrates enzymatic TNF-α release for TIL batch M1030.

FIG. 76 illustrates changes in cell populations of aEM3 cells (C712 (A) and 8B5 (B)) when weaning such cell populations off of FBS to hAB serum media.

FIG. 77 illustrates changes in cell populations of during freeze-thaw-recovery cycles with aEM3 cell populations suspended in various freezing media.

FIG. 78 illustrates the growth of aEM3 cells in gas permeable cell culture flasks over an eight-day time course.

FIG. 79 illustrates a flow panel analysis to determine the purity of aEM3 cells.

FIG. 80 illustrates the results of a flow panel analysis used to determine the purity of aEM3 cells.

FIG. 81 illustrates the differences in cytokine expression between aEM3 feeder cells and PBMC feeders stimulated by OKT3.

FIG. 82 illustrates that TIL may advantageously expanded (pre-REP) with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CM1.

FIG. 83 and FIG. 84 illustrate that TIL may advantageously expanded with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CM1 at Day 11 (PreREP) (FIG. 83) and Day 22 (Pre- and Post-REP) (FIG. 84).

FIG. 85 illustrates that aAPC cells (i.e., aEM3 cells) can be grown and using serum free media. Specifically, CTS OpTimizer and Prime-TCDM were found to be effective in growing aEM3 as compared to cDMEM (10% hSerum). Data shown were mean+SD of five separate experiments. The p value was calculated by the student t-test. *P<0.05.

FIG. 86 and FIG. 87 illustrate the results of two experiments that demonstrate the rapid recovery of aEM3 cells from the TIL-R3 cell line on day 3 following cryopreservation. FIG. 86 illustrates the total cell counts for experiment one and FIG. 87 illustrates the total cell counts for experiment two.

FIG. 88 illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated and grown for 9 days. Cell counts were measured every three days post thaw.

FIG. 89 illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated in GREX 10 flasks and grown for 8 days. Cell counts were measured every four days post thaw.

FIG. 90 illustrates a vector diagram of the pLenti-C-Myc-DDK human OX40L vector.

FIG. 91 illustrates the results of flow cytometry analysis of TILs expanded in a REP with the aEM3 cell line and PBMC feeders, showing that TILs cultured with aEM3 promotes CD8⁺ TIL skewness.

FIG. 92 illustrates the numbers of viable cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.

FIG. 93 illustrates the numbers of CD3⁺ cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.

FIG. 94 illustrates the numbers of CD3⁻ cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.

FIG. 95 illustrates the results of telomere length analysis using a qPCR method.

FIG. 96 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.

FIG. 97 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.

FIG. 98 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is an amino acid sequence for the heavy chain of muromonab.

SEQ ID NO:2 is an amino acid sequence for the light chain of muromonab.

SEQ ID NO:3 is an amino acid sequence for recombinant human IL-2.

SEQ ID NO:4 is an amino acid sequence for aldesleukin.

SEQ ID NO:5 is an amino acid sequence for recombinant human IL-7.

SEQ ID NO:6 is an amino acid sequence for recombinant human IL-15.

SEQ ID NO:7 is an amino acid sequence for recombinant IL-21.

SEQ ID NO:8 is the amino acid sequence of human CD86.

SEQ ID NO:9 is the amino acid sequence of human 4-1BBL (CD137L).

SEQ ID NO:10 is the amino acid sequence of human OX40L (CD134L).

SEQ ID NO:11 is the amino acid sequence of human CD28.

SEQ ID NO:12 is the amino acid sequence of human CTLA-4.

SEQ ID NO:13 is the amino acid sequence of human 4-1BB (CD137).

SEQ ID NO:14 is the amino acid sequence of human OX40 (CD134).

SEQ ID NO:15 is a nucleotide sequence for the pLV430G 4-1BBL empty vector.

SEQ ID NO:16 is a nucleotide sequence for the 4-1BBL CoOP portion of the pLV430G human 4-1BBL vector.

SEQ ID NO:17 is a nucleotide sequence for the 4-1BBL PCRP.

SEQ ID NO:18 is a nucleotide sequence for the pLV430G hCD86 empty vector.

SEQ ID NO:19 is a nucleotide sequence for the hCD86 CoOP portion of the pLV430G human hCD86 vector.

SEQ ID NO:20 is a nucleotide sequence for the hCD86 CoOP B1 B2 PCRP portion of the pLV430G human hCD86 vector.

SEQ ID NO:21 is a nucleotide sequence for the pDONR221 hCD86 vector.

SEQ ID NO:22 is a nucleotide sequence for the pDONR221 4-1BBL vector.

SEQ ID NO:23 is a nucleotide sequence for the pLV430G vector.

SEQ ID NO:24 is a nucleotide sequence for the pDONR221 vector.

SEQ ID NO:25 is a nucleotide sequence for the psPAX2 helper plasmid for lentiviral production.

SEQ ID NO:26 is a nucleotide sequence for the pCIGO-VSV.G helper plasmid for lentiviral production.

SEQ ID NO:27 is the amino acid sequence of the mFc-7C12 scFv clone.

SEQ ID NO:28 is the amino acid sequence of the mFc-8B3 scFv clone.

SEQ ID NO:29 is a nucleotide sequence for the mFC-7C12 scFv.

SEQ ID NO:30 is a nucleotide sequence for the mFC-8B3 scFv.

SEQ ID NO:31 is a nucleotide sequence for the destination vector pLV4301G.

SEQ ID NO:32 is a nucleotide sequence for the donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII L1R5.

SEQ ID NO:33 is a nucleotide sequence for the donor vector 2, pMK hCD8a scaffold TN L5 L2.

SEQ ID NO:34 is a nucleotide sequence for the final vector used for lentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.

SEQ ID NO:35 is a nucleotide sequence for the destination vector, pLV4301G.

SEQ ID NO:36 is a nucleotide sequence for the donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5.

SEQ ID NO:37 is a nucleotide sequence for the donor vector 2, pMK hCD8a scaffold TN L5 L2.

SEQ ID NO:38 is a nucleotide sequence for the final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.

SEQ ID NO:39 is a nucleotide sequence for pLenti-C-Myc-DDK OX40L vector for lentiviral production.

SEQ ID NO:40 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.

SEQ ID NO:41 is a nucleotide sequence for Tel-2b primer used for quantitative polymerase chain reaction measurements of telomere length.

SEQ ID NO:42 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.

SEQ ID NO:43 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

Definitions

The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients to a human subject so that both active pharmaceutical ingredients and/or their metabolites are present in the human subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present is also encompassed in the methods of the invention.

The term “in vivo” refers to an event that takes place in a subject's body.

The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject's body. Aptly, the cell, tissue and/or organ may be returned to the subject's body in a method of surgery or treatment.

The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by major histocompatibility complex (MEW) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.

The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the human subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit in a human subject. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to herein as “freshly harvested” or “a first population of TILs”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”, or “second population of TILs” or “third population of TILs” where appropriate).

TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.

By “cryopreserved TILs” herein is meant that TILs are treated and stored in the range of about −150° C. to −60° C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.

By “population of cells” (including TILs) herein is meant a number of cells that share common traits.

The term “central memory T cell” refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7^(hi)) and CD62L (CD62^(hi)). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.

The term “effector memory T cell” refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7^(lo)) and are heterogeneous or low for CD62L expression (CD62L^(lo)). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.

The terms “sequence identity,” “percent identity,” and “sequence percent identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

The term “conservative amino acid substitutions” means amino acid sequence modifications which do not abrogate the binding of an antibody to an antigen or a protein to its ligand. Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in a 4-1BBL or CD86 protein is preferably replaced with another amino acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen or ligand binding are well-known in the art (see, e.g., Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

The term “retrovirus” refers to RNA viruses that utilize reverse transcriptase during their replication cycle, wherein retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. The double-stranded DNA form is integrated into the chromosome of the infected cell (a “provirus”). The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called “long terminal repeats” or “LTRs.” The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. Several genera included within the family Retroviridae, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, Gammaretrovirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. Because they are integrated into the host DNA, they are capable of transmitting sequences of host DNA from cell to cell. Example gammaretroviral vectors include those derived from the amphotropic Moloney murine leukemia virus (MLV-A), which use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See, e.g., Gordon and Anderson, Curr. Op. Biotechnol., 1994, 5, 611-616 and Miller, et al., Meth. Enzymol., 1993, 217, 581-599, the disclosures of which are incorporated herein by reference.

The term “lentivirus” refers to a genus that includes HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).

The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706.

TABLE 1 Amino acid sequences of muromonab. Identifier (Description) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1 QVQLQQSGAE LARPGASVKM SCHASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY  60 (Muromonab NQHFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120 heavy chain) KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KARGQPREPQ VYTLPPSRDE 360 LTKNQVSLTC LVEGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH  60 (Muromonab FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120 light chain) SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180 TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213

The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Pat. No. 6,706,289, the disclosure of which is incorporated by reference herein.

The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-7 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:5).

The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4⁺ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).

TABLE 2 Amino acid sequences of interleukins. Identifier (Description) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60 (recombinant EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120 human IL-2 RWITFCQSII STLT 134 (rhIL-2)) SEQ ID NO: 4 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60 (aldesleukin) ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120 ITFSQSIIST LT 132 SEQ ID NO: 5 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA  60 (recombinant ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP THSLEENKSL 120 human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153 (rhIL-7)) SEQ ID NO: 6 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60 (recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS 115 human IL-15 (rhIL-15)) SEQ ID NO: 7 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60 (recombinant NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQHMIHQ 120 human IL-21 HLSSRTHGSE DS 132 (rhIL-21))

The term “myeloid cell” as used herein refers to cells of the myeloid lineage or derived therefrom. The myeloid lineage includes a number of morphologically, phenotypically, and functionally distinct cell types including different subsets of granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, erythrocytes, megakaryocytes, and mast cells. In certain embodiments, the myeloid cell is a cell derived from a cell line of myeloid lineage.

“MOLM-14” refers to a human leukemia cell line which was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-A/B/C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86. The MOLM-14 cell line is deposited at DSMZ under Accession No. ACC777. The closely related MOLM-13 cell line is deposited at DSMZ under Accession No. ACC554. As used herein the term “MOLM-14 cell” refers to a MOLM-14 cell and/or a cell derived from the deposited MOLM-14 parental cell line. As used herein the term “MOLM-13 cell” refers to a MOLM-13 cell and/or a cell derived from the deposited MOLM-13 parental cell line.

“EM-3” refers to a human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CIVIL. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33. The EM-3 cell line is deposited at DSMZ under Accession No. ACC134 whilst the closely related EM-2 cell line is deposited at DSMZ under Accession No. ACC135. As used herein the term “EM-3 cell” refers to a EM-3 cell and/or a cell derived from the deposited EM-3 parental cell line.

As used herein, the term “a CD86 protein” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:8 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:8, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

As used herein, the term “4-1BBL” or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:9 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:9, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

As used herein, the term “OX40L” or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:10 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:10, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

The term “biosimilar” means a biological product, including a monoclonal antibody or fusion protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC. The already authorized original biological medicinal product may be referred to as a “reference medicinal product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and/or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and/or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and/or truncation which is/are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and/or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and/or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies.

As used herein, the term “variant” encompasses but is not limited to proteins, antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference protein or antibody by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference protein or antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference protein or antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference protein or antibody. The term “variant” also includes pegylated antibodies or proteins.

“Pegylation” refers to a modified antibody, or a fragment thereof, or protein that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody, antibody fragment, or protein. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody or protein. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C₁-C₁₀) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody or protein to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies and proteins described herein, as described for example in European Patent Nos. EP 0154316 and EP 0401384.

The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

Artificial Antigen Presenting Cells

In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-A/B/C, CD64, CD80, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell that is modified to express one or more costimulatory molecules. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell that is modified to express one or more costimulatory molecules.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell that endogenously expresses HLA-A/B/C, CD64, CD80, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the MOLM-14 cell expresses CD86 and OX40L. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the MOLM-13 cell expresses CD86 and OX40L. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, and conservative amino acid substitutions thereof, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In any of the foregoing embodiments, it will be understood that an aAPC comprising a MOLM-14 or MOLM-13 cell may be modified to express both OX40L and 4-1BBL.

The sequences for human CD86, human 4-1BBL (CD137L), and human OX40L (CD134L) are given in Table 3.

TABLE 3 Amino acid sequences for human CD86, human 4-1BBL, and human OX40L. Identifier (Description) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 8 MGLSNILFVM AFLLSGAAPL KIQAYFNETA DLPCQFANSQ NQSLSELVVF WQDQENLVLN  60 (human CD86) EVYLGKEKED SVHSKYMGRT SFDSDSWTLR LHNLQIKDKG LYQCIIHHKK PTGMIRIHQM 120 NSELSVLANF SQPEIVPISN ITENVYINLT CSSIHGYPEP KKMSVLLRTK NSTIEYDGIM 180 QESQDNVTEL YDVSISLSVS FPDVTSNMTI FCILETDKTR LLSSPFSIEL EDPQPPPDHI 240 PWITAVLPTV IICVMVFCLI LWKWKKKKRP RNSYKCGTNT MEREESEQTK KREKIHIPER 300 SDEAQRVFKS SKTSSCDKSD TCF 323 SEQ ID NO: 9 MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA  60 (human 4-1BBL, SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL 120 CD137) TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA 180 LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV 240 TPEIPAGLPS PRSE 254 SEQ ID NO: 10 MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL QVSHRYPRIQ  60 (human OX40L, SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVNISLHYQ 120 CD134L) KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF 180 CVL 183

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

The sequences for the ligands to which human CD86 binds (CD28 and CTLA-4), the ligand to which human 4-1BBL binds (4-1BB), and the ligand to which human OX40L binds (OX40) are given in Table 4.

TABLE 4 Amino acid sequences for human CD28, human CTLA-4, human 4-1BB, and human OX40. Identifier (Description) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 11 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD  60 (human CD28) SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 120 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 180 SHRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS 220 SEQ ID NO: 12 MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY  60 (human CTLA-4) ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR 120 AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL 180 LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN 223 SEQ ID NO: 13 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR  60 (human 4-1BB) TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 120 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG THERDVVCGP SPADLSPGAS SVTPPAPARE 180 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 240 CSCRFPEEEE GGCEL 255 SEQ ID NO: 14 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ  60 (human OX40) NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 180 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 240 RRDQRLPPDA FKPPGGGSFR TPIQEEQADA HSTLAKI 277

In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-A/B/C, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from an EM-3 parental cell line. In an embodiment, the invention includes an aAPC comprising an EM-3 cell that is modified to express one or more costimulatory molecules. In an embodiment, the invention includes an aAPC comprising an EM-2 cell that is modified to express one or more costimulatory molecules.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell that expresses HLA-A/B/C, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 96. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 97. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell that expresses HLA-A/B/C, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the EM-3 cell expresses CD86 and OX40L. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 96. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 97. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98.

In any of the foregoing embodiments, it is understood that an aAPC comprising an EM-3 or EM-2 cell may be modified to express both OX40L and 4-1BBL.

In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from a K562-lineage parental cell line. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell that is modified to express one or more costimulatory molecules. In an embodiment, the K562 lineage parental cell line is deposited under accession no. ATCC CCL-243 and also at European Collection of Authenticated Cell Cultures (ECACCECACC 89121407).

In an embodiment, the invention includes an aAPC comprising a K562-lineage cell that expresses CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.

In an embodiment, the invention includes an aAPC comprising a K562-lineage cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the K562-lineage cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:11, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an K562-lineage cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.

Methods of Preparing Artificial Antigen Presenting Cells

In an embodiment, a method of preparing an aAPC includes the step of stable incorporation of genes for production of CD86 and 4-1BBL. In an embodiment, a method of preparing an aAPC includes the step of retroviral transduction. In an embodiment, a method of preparing an aAPC includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of gamma-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated by reference herein.

In an embodiment, a method of preparing an aAPC includes the step of stable incorporation of genes for transient production of CD86 and 4-1BBL. In an embodiment, a method of preparing an aAPC includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J. 1991, 60, 297-306, and U.S. Patent Application Publication No. 2014/0227237 A1, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and in U.S. Pat. No. 5,593,875, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Pat. Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of transfection using methods described in U.S. Pat. Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

In an embodiment, the aAPC is transduced by first using the Gateway cloning method (commercially available from ThermoFisher, Inc.) to prepare vector for lentiviral transduction, followed by lentiviral transduction using the vector and one or more associated helper plasmids, as is also described elsewhere herein. In the Gateway cloning method, a gene is selected (such as CD86) and is then provided with primers and amplified using PCR technology with the help of an attB tagged primer pair. The PCR fragment is then combined with a donor vector (pDONR, such as pDONR221) that includes attP sites to provide an entry clone, using the BP reaction. An integration reaction between the attB and the attP sites combines the PCR fragment with the donor vector. The resulting entry clone contains the gene of interest flanked by attL sites. The LR reaction is then used to combine the entry clone with a destination vector to produce an expression vector. In the LR reaction, a recombination reaction is used to link the entry clone with the destination vector (such as pLV430G) using the attL and attR sites and a clonase enzyme. The attL sites are already found in the entry clone, while the destination vector includes the attR sites. The LR reaction is carried out to transfer the sequence of interest into one or more destination vectors in simultaneous reactions.

In some embodiments, the aAPCs described herein may be grown and maintained under serum-based media and/or serum free media. According to an exemplary method, aAPCs may be cultured in 24 well plates at a cell density of about 1×10⁶ cells per well for 3 to 5 days. The cells may then be isolated and/or washed by centrifugation and resuspended in media or cryopreserved in an appropriate cryopreservation media (e.g., CryoStor 10 (BioLife Solutions)) and stored in a −80° C. freezer.

In some embodiments, the aAPCs described herein may be grown in the presence of serum-based media. In some embodiments, the aAPCs described herein by may be grown in the presence of serum-based media that includes human serum (hSerum) containing media (e.g., cDMEM with 10% hSerum). In some embodiments, the aAPCs grown in the presence of serum-based media may be selected from the group consisting of aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.

In some embodiments, the aAPCs described herein may be grown in the presence of serum free media. In some embodiments, the serum free media may be selected from the group consisting of CTS Optmizer (ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), XFSM (MesenCult), and the like. In some embodiments, the aAPCs grown in the presence of serum free media may be selected from the group consisting of aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.

Methods of Expanding Tumor Infiltrating Lymphocytes and T Cells

In an embodiment, the invention includes a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising at least one TIL with an aAPC described herein, wherein said aAPC comprises at least one co-stimulatory ligand that specifically binds with a co-stimulatory molecule expressed on the cellular surface of the TILs, wherein binding of said co-stimulatory molecule with said co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs) using any of the aAPCs of the present disclosure, the method comprising the steps as described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated by reference herein. For example, the tumor may be placed in enzyme media and mechanically dissociated for approximately 1 minute. The mixture may then be incubated for 30 minutes at 37° C. in 5% CO₂ and then mechanically disrupted again for approximately 1 minute. After incubation for 30 minutes at 37° C. in 5% CO₂, the tumor may be mechanically disrupted a third time for approximately 1 minute. If after the third mechanical disruption, large pieces of tissue are present, 1 or 2 additional mechanical dissociations may be applied to the sample, with or without 30 additional minutes of incubation at 37° C. in 5% CO₂. At the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll may be performed to remove these cells. TIL cultures were initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, N.Y.), each well may be seeded with 1×10⁶ tumor digest cells or one tumor fragment approximately 1 to 8 mm³ in size in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. Cultures may be initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm² gas-permeable silicon bottom (G-Rex 10; Wilson Wolf Manufacturing, New Brighton, each flask may be loaded with 10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2. G-Rex 10 and 24-well plates may be incubated in a humidified incubator at 37° C. in 5% CO₂ and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be changed every 2-3 days. Rapid expansion protocol (REP) of TILs may be performed using T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks, as described elsewhere herein, using the aAPCs of the present disclosure. For REP in T-175 flasks, 1×10⁶ TILs may be suspended in 150 mL of media in each flask. The TIL may be cultured with aAPCs of the present disclosure at a ratio described herein, in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The T-175 flasks may be incubated at 37° C. in 5% CO₂. Half the media may be changed on day 5 using 50/50 medium with 3000 IU/mL of IL-2. On day 7, cells from 2 T-175 flasks may be combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may be added to the 300 mL of TIL suspension. The number of cells in each bag may be counted every day or two days, and fresh media may be added to keep the cell count between 0.5 and 2.0×10⁶ cells/mL. For REP in 500 mL capacity flasks with 100 cm² gas-permeable silicon bottoms (e.g., G-Rex 100, Wilson Wolf Manufacturing, as described elsewhere herein), 5×10⁶ or 10×10⁶ TILs may be cultured with aAPCs at a ratio described herein (e.g., 1 to 100) in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO₂. On day five, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The obtained TIL pellets may be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2 and added back to the G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, on day seven the TIL in each G-Rex100 are suspended in the 300 mL of media present in each flask and the cell suspension may be divided into three 100 mL aliquots that may be used to seed 3 G-Rex100 flasks. About 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may then be added to each flask. G-Rex100 flasks may then be incubated at 37° C. in 5% CO₂, and after four days, 150 mL of AIM-V with 3000 IU/mL of IL-2 may be added to each G-Rex100 flask. After this, the REP may be completed by harvesting cells on day 14 of culture.

As described herein, TILs may be expanded advantageously in the presence of serum free media. In some embodiments, the TIL expansion methods described herein may include the use of serum free media rather than serum-based media (e.g., complete media or CM1). In some embodiments, the TIL expansion methods described herein may use serum free media rather than serum-based media. In some embodiments, the serum free media may be selected from the group consisting of CTS Optmizer (ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), and the like.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the cell culture medium further comprises IL-2 at an         initial concentration of about 3000 IU/mL and OKT-3 antibody at         an initial concentration of about 30 ng/mL.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the population of APCs expands the population of TILs by         at least 50-fold over a period of 7 days in a cell culture         medium.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the myeloid cell endogenously expresses HLA-A/B/C,         ICOS-L, and CD58.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the myeloid cell is a MOLM-14 cell.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the myeloid cell is a MOLM-13 cell.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (c) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (d) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the myeloid cell is a EM-3 cell.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the CD86 protein comprises an amino acid sequence as set         forth in SEQ ID NO:8, or conservative amino acid substitutions         thereof, and the 4-1BBL protein comprises an amino acid sequence         as set forth in SEQ ID NO:9, or conservative amino acid         substitutions thereof.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the nucleic acid encoding CD86 comprises a nucleic acid         sequence as set forth in SEQ ID NO:19 and the nucleic acid         encoding 4-1BBL comprises a nucleic acid sequence as set forth         in SEQ ID NO:16.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the expansion is performed using a gas permeable         container.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the ratio of the population of TILs to the population of         aAPCs is between 1 to 200 and 1 to 400.

In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to         obtain a population of artificial antigen presenting cells         (aAPCs), wherein the one or more viral vectors comprise a         nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,         and wherein the myeloid cell expresses a CD86 protein and a         4-1BBL protein, and     -   (b) contacting the population of TILs with the population of         aAPCs in a cell culture medium,     -   wherein the ratio of the population of TILs to the population of         aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a myeloid artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co-stimulatory molecule on the TILs, wherein binding of the co-stimulatory molecules with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least two co-stimulatory ligands comprise CD86 and 4-1BBL.

In any of the foregoing embodiments, the aAPC may further comprise OX40L in addition to 4-1BBL, or may comprise OX40L instead of 4-1BBL.

In an embodiment, a method of expanding or treating a cancer includes a step wherein TILs are obtained from a patient tumor sample. A patient tumor sample may be obtained using methods known in the art. For example, TILs may be cultured from enzymatic tumor digests and tumor fragments (about 1 to about 8 mm³ in size) from sharp dissection. Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37° C. in 5% CO₂, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.

In an embodiment, REP can be performed in a gas permeable container using the aAPCs of the present disclosure by any suitable method. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T cell receptor stimulus can include, for example, about 30 ng/mL of an anti-CD3 antibody, e.g. OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, N.J., USA or Miltenyi Biotech, Auburn, Calif., USA) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA). TILs can be rapidly expanded by further stimulation of the TILs in vitro with one or more antigens, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T cell growth factor, such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, a method for expanding TILs may include using about 5000 mL to about 25000 mL of cell culture medium, about 5000 mL to about 10000 mL of cell culture medium, or about 5800 mL to about 8700 mL of cell culture medium. In an embodiment, a method for expanding TILs may include using about 1000 mL to about 2000 mL of cell medium, about 2000 mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000 mL of cell culture medium, about 4000 mL to about 5000 mL of cell culture medium, about 5000 mL to about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL to about 8000 mL of cell culture medium, about 8000 mL to about 9000 mL of cell culture medium, about 9000 mL to about 10000 mL of cell culture medium, about 10000 mL to about 15000 mL of cell culture medium, about 15000 mL to about 20000 mL of cell culture medium, or about 20000 mL to about 25000 mL of cell culture medium. In an embodiment, expanding the number of TILs uses no more than one type of cell culture medium. Any suitable cell culture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad, Calif., USA). In this regard, the inventive methods advantageously reduce the amount of medium and the number of types of medium required to expand the number of TIL. In an embodiment, expanding the number of TIL may comprise feeding the cells no more frequently than every third or fourth day. Expanding the number of cells in a gas permeable container simplifies the procedures necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells.

In an embodiment, the rapid expansion is performed using a gas permeable container. Such embodiments allow for cell populations to expand from about 5×10⁵ cells/cm² to between 10×10⁶ and 30×10⁶ cells/cm². In an embodiment, this expansion occurs without feeding. In an embodiment, this expansion occurs without feeding so long as medium resides at a height of about 10 cm in a gas-permeable flask. In an embodiment this is without feeding but with the addition of one or more cytokines. In an embodiment, the cytokine can be added as a bolus without any need to mix the cytokine with the medium. Such containers, devices, and methods are known in the art and have been used to expand TILs, and include those described in U.S. Patent Application Publication No. US 2014/0377739 A1, International Patent Application Publication No. WO 2014/210036 A1, U.S. Patent Application Publication No. US 2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228 A1, U.S. Pat. No. 8,809,050, International Patent Application Publication No. WO 2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216 A1, U.S. Patent Application Publication No. US 2012/0244133 A1, International Patent Application Publication No. WO 2012/129201 A1, U.S. Patent Application Publication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860, International Patent Application Publication No. WO 2013/173835 A1, and U.S. Patent Application Publication No. US 2015/0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated by reference herein.

In an embodiment, the gas permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 10 cm² gas permeable culture surface. In an embodiment, the gas permeable container includes a 40 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 100 to 300 million TILs after 2 medium exchanges.

In an embodiment, the gas permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm² gas permeable culture surface. In an embodiment, the gas permeable container includes a 450 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs after 2 medium exchanges.

In an embodiment, the gas permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm² gas permeable culture surface. In an embodiment, the gas permeable container includes a 1000 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs without medium exchange.

In an embodiment, the gas permeable container is a G-Rex 100 L flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm² gas permeable culture surface. In an embodiment, the gas permeable container includes a 2000 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs without medium exchange.

In an embodiment, the gas permeable container is a G-Rex 24 well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 2 cm² gas permeable culture surface. In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 8 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 20 to 60 million cells per well after 2 medium exchanges.

In an embodiment, the gas permeable container is a G-Rex 6 well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 10 cm² gas permeable culture surface. In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 40 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 100 to 300 million cells per well after 2 medium exchanges.

In an embodiment, the cell medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells. In an embodiment, the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium therein; obtaining TILs from the tumor tissue sample; expanding the number of TILs in a second gas permeable container containing cell medium therein using aAPCs for a duration of about 14 to about 42 days, e.g., about 28 days.

In an embodiment, the rapid expansion uses about 1×10⁹ to about 1×10¹¹ aAPCs. In an embodiment, the rapid expansion uses about 1×10⁹ aAPCs. In an embodiment, the rapid expansion uses about 1×10¹⁰ aAPCs. In an embodiment, the rapid expansion uses about 1×10¹¹ aAPCs.

In an embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is selected from the group consisting of 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:225, 1:250, 1:275, 1:300, 1:350, 1:400, 1:450, and 1:500. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:90. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:95. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:100. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:105. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:110.

In an embodiment, the ratio of TILs to aAPCs in the rapid expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILs to aAPCs in the rapid expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of TILs to aAPCs in the rapid expansion is between 1 to 100 and 1 to 200.

In an embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises an OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody.

In an embodiment, a rapid expansion process for TILs may be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). For TIL rapid expansion in T-175 flasks, 1×10⁶ TILs suspended in 150 mL of media may be added to each T-175 flask. The TILs may be cultured with aAPCs at a ratio of 1 TIL to 100 aAPCs and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU (international units) per mL of IL-2 and 30 ng per ml of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks may be incubated at 37° C. in 5% CO₂. Half the media may be exchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. On day 7 cells from two T-175 flasks may be combined in a 3 liter bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0×10⁶ cells/mL.

In an embodiment, for TIL rapid expansions in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×10⁶ or 10×10⁶ TIL may be cultured with aAPCs at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT-3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO₂. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (revolutions per minute; 491×g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-Rex 100 flask. The cells may be harvested on day 14 of culture.

In an embodiment, TILs may be prepared as follows. 2 mm³ tumor fragments are cultured in complete media (CM) comprised of AIM-V medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, Va.), 100 U/mL penicillin (Invitrogen Life Technologies), 100 μg/mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, Va.) and 600 IU/mL rhIL-2 (Chiron, Emeryville, Calif.). For enzymatic digestion of solid tumors, tumor specimens were diced into RPMI-1640, washed and centrifuged at 800 rpm for 5 minutes at 15-22° C., and resuspended in enzymatic digestion buffer (0.2 mg/mL Collagenase and 30 units/ml of DNase in RPMI-1640) followed by overnight rotation at room temperature. TILs established from fragments may be grown for 3-4 weeks in CM and expanded fresh or cryopreserved in heat-inactivated HAB serum with 10% dimethylsulfoxide (DMSO) and stored at −180° C. until the time of study. Tumor associated lymphocytes (TAL) obtained from ascites collections were seeded at 3×10⁶ cells/well of a 24 well plate in CM. TIL growth was inspected about every other day using a low-power inverted microscope.

In an embodiment, TILs are expanded in gas-permeable containers. Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2005/0106717 A1, the disclosures of which are incorporated herein by reference. In an embodiment, TILs are expanded in gas-permeable bags. In an embodiment, TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In an embodiment, TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L. In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 50 and 150 mL, between 150 and 250 mL, between 250 and 350 mL, between 350 and 450 mL, between 450 and 550 mL, between 550 and 650 mL, between 650 and 750 mL, between 750 and 850 mL, between 850 and 950 mL, and between 950 and 1050 mL. In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 1 L and 2 L, between 2 L and 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L, between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9 L and 10 L, between 10 L and 11 L, between 11 L and 12 L, between 12 L and 13 L, between 13 L and 14 L, between 14 L and 15 L, between 15 L and 16 L, between 16 L and 17 L, between 17 L and 18 L, between 18 L and 19 L, and between 19 L and 20 L. In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 0.5 L and 5 L, between 5 L and 10 L, between 10 L and 15 L, between 15 L and 20 L, between 20 L and 25 L, and between 25 L and 30 L. In an embodiment, the cell expansion system utilizes a rocking time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In an embodiment, the cell expansion system utilizes a rocking time of between 30 minutes and 1 hour, between 1 hour and 12 hours, between 12 hours and 1 day, between 1 day and 7 days, between 7 days and 14 days, between 14 days and 21 days, and between 21 days and 28 days. In an embodiment, the cell expansion system utilizes a rocking rate of about 2 rocks/minute, about 5 rocks/minute, about 10 rocks/minute, about 20 rocks/minute, about 30 rocks/minute, and about 40 rocks/minute. In an embodiment, the cell expansion system utilizes a rocking rate of between 2 rocks/minute and 5 rocks/minute, 5 rocks/minute and 10 rocks/minute, 10 rocks/minute and 20 rocks/minute, 20 rocks/minute and 30 rocks/minute, and 30 rocks/minute and 40 rocks/minute. In an embodiment, the cell expansion system utilizes a rocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In an embodiment, the cell expansion system utilizes a rocking angle of between 2° and 3°, between 3° and 4°, between 4° and 5°, between 5° and 6°, between 6° and 7°, between 7° and 8°, between 8° and 9°, between 9° and 10°, between 10° and 11°, and between 11° and 12°.

In an embodiment, a method of expanding TILs using aAPCs further comprises a step wherein TILs are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No. 2016/0010058 A1, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity.

In an embodiment, the aAPCs of the present invention may be used to expand T cells. Any of the foregoing embodiments of the present invention described for the expansion of TILs may also be applied to the expansion of T cells. In an embodiment, the aAPCs of the present invention may be used to expand CD8⁺ T cells. In an embodiment, the aAPCs of the present invention may be used to expand CD4⁺ T cells. In an embodiment, the aAPCs of the present invention may be used to expand T cells transduced with a chimeric antigen receptor (CAR-T). In an embodiment, the aAPCs of the present invention may be used to expand T cells comprising a modified T cell receptor (TCR). The CAR-T cells may be targeted against any suitable antigen, including CD19, as described in the art, e.g., in U.S. Pat. Nos. 7,070,995; 7,446,190; 8,399,645; 8,916,381; and 9,328,156; the disclosures of which are incorporated by reference herein. The modified TCR cells may be targeted against any suitable antigen, including NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof, as described in the art, e.g., in U.S. Pat. Nos. 8,367,804 and 7,569,664, the disclosures of which are incorporated by reference herein.

Methods of Treating Cancers and Other Diseases

The compositions and methods described herein can be used in a method for treating diseases. In an embodiment, they are for use in treating hyperproliferative disorders. They may also be used in treating other disorders as described herein and in the following paragraphs. The TILs, populations and compositions thereof described herein may be for use in the treatment of a disease. In an embodiment, the TILs, populations and compositions described herein are for use in the treatment of a hyperproliferative disorder.

In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma, pancreatic cancer, and glioblastoma. In some embodiments, the hyperproliferative disorder is a hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

In an embodiment, the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing a rapid expansion of the first population of TILs using a population of artificial antigen presenting cells (aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs; and (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer. In an embodiment, the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the rapid expansion is performed over a period not greater than 14 days.

In an embodiment, the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs using a first population of artificial antigen presenting cells (aAPCs) in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 10-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a second population of aAPCs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the first population of TILs; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer. In an embodiment, the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the rapid expansion is performed over a period not greater than 14 days. In an embodiment, the initial expansion is performed using a gas permeable container.

In an embodiment, the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 10-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a population of artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the first population of TILs; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer. In an embodiment, the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the rapid expansion is performed over a period not greater than 14 days.

In an embodiment, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m²/d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Efficacy of the compounds and combinations of compounds described herein in treating, preventing and/or managing the indicated diseases or disorders can be tested using various models known in the art, which provide guidance for treatment of human disease. For example, models for determining efficacy of treatments for ovarian cancer are described, e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, et al., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy of treatments for pancreatic cancer are described in Herreros-Villanueva, et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models for determining efficacy of treatments for breast cancer are described, e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models for determining efficacy of treatments for melanoma are described, e.g., in Damsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Models for determining efficacy of treatments for lung cancer are described, e.g., in Meuwissen, et al., Genes & Development, 2005, 19, 643-664. Models for determining efficacy of treatments for lung cancer are described, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.

Non-Myeloablative Lymphodepletion with Chemotherapy

In an embodiment, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure. In an embodiment, the invention provides a population of TILs obtainable by a method described herein for use in treating a cancer, wherein the population of TILs is for treating a patient which is pre-treated with non-myeloablative chemotherapy. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m²/d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the aAPC-expanded TILs of the invention.

In general, lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated by reference herein in their entireties.

In some embodiments, the fludarabine is administered at a concentration of 0.5 μg/mL-10 μg/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 μg/mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine is administered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 2-7 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 μg/ml-10 μg/ml by administration of cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 μg/mL by administration of cyclophosphamide. In some embodiments, the cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the cyclophosphamide is administered at a dosage of 100 mg/m²/day, 150 mg/m²/day, 175 mg/m²/day 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day, 275 mg/m²/day, or 300 mg/m²/day. In some embodiments, the cyclophosphamide is administered intravenously (i.v.) In some embodiments, the cyclophosphamide treatment is administered for 2-7 days at 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment is administered for 4-5 days at 250 mg/m²/day i.v. In some embodiments, the cyclophosphamide treatment is administered for 4 days at 250 mg/m²/day i.v.

In some embodiments, lymphodepletion is performed by administering the fludarabine and the cyclophosphamide are together to a patient. In some embodiments, fludarabine is administered at 25 mg/m²/day i.v. and cyclophosphamide is administered at 250 mg/m²/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg/m²/day for two days followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using aAPCs of the present disclosure are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using aAPCs of the present disclosure may be administered by any suitable route as known in the art. Preferably, the TILs are administered as a single infusion, such as an intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. Preferably, from about 2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an average of around 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In an embodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.

In some embodiments, the number of the TILs provided in the pharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 2×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of the TILs provided in the pharmaceutical compositions of the invention is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

In some embodiments, the amount of the TILs provided in the pharmaceutical compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

The TILs provided in the pharmaceutical compositions of embodiments of the invention are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹² 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effective dosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.

EXAMPLES

The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example 1—Variability in Expansion of Tumor Infiltrating Lymphocytes Using PBMC Feeder Cells

The variability in TIL expansion obtained by use of PBMC feeder cells may be demonstrated by comparing the results of multiple TIL expansions on the same line of TILs obtained from a patient. FIG. 1 illustrates typical results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells (PBMC feeders). Two TIL lines labeled M1015T and M1016T (1.3×10⁵ cells) were co-cultured with 46 different irradiated feeder cell lots (1.3×10⁷), IL-2 (3000 IU/mL, recombinant human IL-2 (e.g., aldesleukin or equivalent), CellGenix, Inc., Portsmouth, N.H., USA) and OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in a T25 flask for 7 days. The fold expansion value for TILs was calculated on Day 7. The figure shows the number of fold expansions for the two TIL lines in separate stimulation experiments. For each TIL line, 46 different PBMC feeder lots were tested. The results range over more than 100-fold for each TIL line, and highlight the variability of expansion results using PBMC feeder cells. The aAPCs of the present invention offer reduced variability in expansion performance compared to PBMC feeders, as well as other advantages, as shown in the following examples.

Example 2—Selection of Myeloid Cells for aAPC Development

Phenotypic characterization was performed on various myeloid-lineage cell lines to identify potential candidates for further modification into aAPCs for TIL expansion. The results are summarized in Table 5. The MOLM-14 cell line exhibited endogenous expression of CD64, and was selected for further development. The EM-3 cell line was selected based on the observation of endogenous expression of ICOS-L (which was not observed for the EM-2 cell line, despite being taken from the same patient).

TABLE 5 Summary of costimulatory molecules expressed endogenously on candidate cell lines for aAPCs. CML refers to chronic myeloid leukemia, and AML refers to acute myeloid leukemia. “Pop” refers to the population of cells observed to express the marker (½ pop = 50%). Cell line K562 myeloid EM-2 EM-3 erythro- Myeloid blast Myeloid blast KG1-246 KG1-8031 leukemia, MOLM-14 Origin crisis, CML crisis, CML AML AML CML AML HLA-A/B/C + + + + − + CD64 − − − − − + CD80 − − − − − + ICOS-L − + − − − + 4-1BBL − − − − − − PD-L1 − − − − − − CD58 + + + + + + CD86 − − − − − + (½ pop)

Example 3—Preparation of MOLM-14 Artificial Antigen Presenting Cells (aMOLM14 aAPCs)

MOLM-14 cells were obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH. To develop MOLM-14 based aAPCs, MOLM-14 cells were engineered with the costimulatory molecules CD86 and 4-1BBL (CD137L). Human CD86 (hCD86) and human 4-1BBL (h4-1BBL) genes were cloned into commercially-available PLV430G and co-transfected with PDONR221 vectors (Invitrogen/Thermo Fisher Scientific, Carlsbad, Calif., USA) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4, 571-589, to clone hCD86 and hCD137L genes onto the PLV430G and PDONR221 vectors. The 293T cell line (human embryonic kidney cells transformed with large T antigen) was used for lentiviral production, transduced to MOLM-14 cells. The transfected cells were sorted (S3e Cell Sorter, Bio-Rad, Hercules, Calif., USA) using APC-conjugated CD86 and PE-conjugated CD137L to isolate and enrich the cells. The enriched cells were checked for purity by flow cytometry.

The vectors and portions thereof used for cloning are depicted in FIG. 2 to FIG. 11, and the nucleotide sequences for each vector are given in Table 6. The pLV430G human 4-1BBL vector is illustrated in FIG. 2, with the polymerase chain reaction product (PCRP) portion shown in FIG. 3. The pLV430G human CD86 vector is illustrated in FIG. 4, with the PCRP portion shown in FIG. 5. The pDONR221 human CD86 donor and human 4-1BBL donor vectors are shown in FIG. 6 and FIG. 7, respectively. Diagrams of the empty pLV430G destination vector and empty pDONR221 donor vector for the Gateway cloning method are shown in FIG. 8 and FIG. 9, respectively. FIG. 10 and FIG. 11 illustrate vector diagrams of the psPAX2 and pCIGO-VSV.G helper plasmids used for lentivirus production.

TABLE 6 Nucleotide sequences for preparation of lentivirus for transduction of aAPCs. Identifier (Description) Sequence SEQ ID NO: 15 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg    60 (pLV430G human gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac   120 4-1BBL vector) cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat   180 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc   240 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac   300 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct   360 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc   420 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac   480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat   540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg   600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc   660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct   720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg   780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg   840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag   900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat   960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag  1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca  1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata  1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga  1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc  1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata  1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag  1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag  1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat  1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc  1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa  1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca  1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc  1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct  1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata  1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat  1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag  1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga  2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat  2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg  2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa  2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa  2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg  2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag  2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag  2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc  2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca  2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc  2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat  2700 caacaagttt gtacaaaaaa gcaggcttcg ccaccatgga atacgcctct gatgccagcc  2760 tggaccccga agctccttgg cctcctgccc ctagagccag agcctgtaga gtgctgcctt  2820 gggctctggt ggctggcctt ctccttctgc tgctgctggc cgctgcctgc gctgtgtttc  2880 tggcttgtcc ttgggccgtg tcaggcgcca gagcttctcc tggatctgcc gccagcccca  2940 gactgagaga gggacctgag ctgagccccg atgatcctgc cggactgctg gatctgagac  3000 agggcatgtt cgcccagctg gtggcccaga acgtgctgct gatcgatggc cccctgagct  3060 ggtacagcga tcctggactg gctggcgtgt cactgacagg cggcctgagc tacaaagagg  3120 acaccaaaga actggtggtg gccaaggccg gcgtgtacta cgtgttcttt cagctggaac  3180 tgcggagagt ggtggccggc gaaggatccg gctctgtgtc tctggcactg catctgcagc  3240 ccctgagatc tgctgcaggc gctgctgcac tggccctgac agtggacctg cctccagcct  3300 ctagcgaggc cagaaactcc gcattcgggt ttcaaggcag actgctgcac ctgtctgccg  3360 gccagagact gggagtgcat ctgcacacag aggccagagc cagacacgcc tggcagctga  3420 cacagggcgc tacagtgctg ggcctgttca gagtgacccc cgaaattcca gccggcctgc  3480 ccagccctag aagcgagtag gacccagctt tcttgtacaa agtggtgatt cgagttaatt  3540 aagctagcct agtgccattt gttcagtggt tcgtagggct ttcccccact gtttggcttt  3600 cagttatatg gatgatgtgg tattgggggc caagtctgta cagcatcttg agtccctttt  3660 taccgctgtt accaattttc ttttgtcttt gggtatacat ttaaacccta acaaaacaaa  3720 gagatggggt tactctctaa attttatggg ttatgtcatt ggatgttatg ggtccttgcc  3780 acaagaacac atcatacaaa aaatcaaaga atgttttaga aaacttccta ttaacaggcc  3840 tattgattgg aaagtatgtc aacgaattgt gggtcttttg ggttttgctg ccccttttac  3900 acaatgtggt tatcctgcgt tgatgccttt gtatgcatgt attcaatcta agcaggcttt  3960 cactttctcg ccaacttaca aggcctttct gtgtaaacaa tacctgaacc tttaccccgt  4020 tgcccggcaa cggccaggtc tgtgccaagt gtttgctgac gcaaccccca ctggctgggg  4080 cttggtcatg ggccatcagc gcatgcgtgg aaccttttcg gctcctctgc cgatccatac  4140 tgcggaactc ctagccgctt gttttgctcg cagcaggtct ggagcaaaca ttatcgggac  4200 tgataactct gttgtcctat cccgcaaata tacatcgttt ccatggctgc taggctgtgc  4260 tgccaactgg atcctgcgcg ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc  4320 ggacgaccct tctcggggtc gcttgggact ctctcgtccc cttctccgtc tgccgttccg  4380 accgaccacg gggcgcacct ctctttacgc ggactccccg tctgtgcctt ctcatctgcc  4440 ggaccgtgtg cacttcgctt cacctctgca cgtcgcatgg agaccaccgt gaacgcccac  4500 caaatattgc ccaaggtctt acataagagg actcttggac tctcagcaat gtcaacgacc  4560 gaccttgagg catacttcaa agactgtttg tttaaagact gggaggagtt gggggaggag  4620 attaggttaa aggtctttgt actaggaggc tgtaggcata aattggtctg cgcaccagca  4680 ccatggcgca atcactagag cggggtacct ttaagaccaa tgacttacaa ggcagctgta  4740 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga  4800 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg  4860 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg  4920 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc  4980 ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat  5040 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat  5100 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg  5160 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta  5220 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc  5280 cccatggctg actaattttt tttatttatg cagaggccga ggccggatcc cttgagtggc  5340 tttcatcctg gagcagactt tgcagtctgt ggactgcaac acaacattgc ctttatgtgt  5400 aactcttggc tgaagctctt acaccaatgc tgggggacat gtacctccca ggggcccagg  5460 aagactacgg gaggctacac caacgtcaat cagaggggcc tgtgtagcta ccgataagcg  5520 gaccctcaag agggcattag caatagtgtt tataaggccc ccttgttaat tcttgaagac  5580 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt  5640 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct  5700 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat  5760 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg  5820 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg  5880 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc  5940 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat  6000 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact  6060 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca  6120 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact  6180 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg  6240 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg  6300 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg  6360 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg  6420 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag  6480 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc  6540 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga  6600 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat  6660 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc  6720 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag  6780 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct  6840 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac  6900 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc  6960 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg  7020 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt  7080 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt  7140 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc  7200 attgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca  7260 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata  7320 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg  7380 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct  7440 ggcctttttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca  7500 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc  7560 ctcgcgtcg  7569 SEQ ID NO: 16 atggaatacg cctctgatgc cagcctggac cccgaagctc cttggcctcc tgcccctaga    60 (4-1BBL CoOP) gccagagcct gtagagtgct gccttgggct ctggtggctg gccttctcct tctgctgctg   120 ctggccgctg cctgcgctgt gtttctggct tgtccttggg ccgtgtcagg cgccagagct   180 tctcctggat ctgccgccag ccccagactg agagagggac ctgagctgag ccccgatgat   240 cctgccggac tgctggatct gagacagggc atgttcgccc agctggtggc ccagaacgtg   300 ctgctgatcg atggccccct gagctggtac agcgatcctg gactggctgg cgtgtcactg   360 acaggcggcc tgagctacaa agaggacacc aaagaactgg tggtggccaa ggccggcgtg   420 tactacgtgt tctttcagct ggaactgcgg agagtggtgg ccggcgaagg atccggctct   480 gtgtctctgg cactgcatct gcagcccctg agatctgctg caggcgctgc tgcactggcc   540 ctgacagtgg acctgcctcc agcctctagc gaggccagaa actccgcatt cgggtttcaa   600 ggcagactgc tgcacctgtc tgccggccag agactgggag tgcatctgca cacagaggcc   660 agagccagac acgcctggca gctgacacag ggcgctacag tgctgggcct gttcagagtg   720 acccccgaaa ttccagccgg cctgcccagc cctagaagcg agtag   765 SEQ ID NO: 17 ggggacaagt ttgtacaaaa aagcaggctt cgccaccatg gaatacgcct ctgatgccag    60 (4-1BBL PRCP) cctggacccc gaagctcctt ggcctcctgc ccctagagcc agagcctgta gagtgctgcc   120 ttgggctctg gtggctggcc ttctccttct gctgctgctg gccgctgcct gcgctgtgtt   180 tctggcttgt ccttgggccg tgtcaggcgc cagagcttct cctggatctg ccgccagccc   240 cagactgaga gagggacctg agctgagccc cgatgatcct gccggactgc tggatctgag   300 acagggcatg ttcgcccagc tggtggccca gaacgtgctg ctgatcgatg gccccctgag   360 ctggtacagc gatcctggac tggctggcgt gtcactgaca ggcggcctga gctacaaaga   420 ggacaccaaa gaactggtgg tggccaaggc cggcgtgtac tacgtgttct ttcagctgga   480 actgcggaga gtggtggccg gcgaaggatc cggctctgtg tctctggcac tgcatctgca   540 gcccctgaga tctgctgcag gcgctgctgc actggccctg acagtggacc tgcctccagc   600 ctctagcgag gccagaaact ccgcattcgg gtttcaaggc agactgctgc acctgtctgc   660 cggccagaga ctgggagtgc atctgcacac agaggccaga gccagacacg cctggcagct   720 gacacagggc gctacagtgc tgggcctgtt cagagtgacc cccgaaattc cagccggcct   780 gcccagccct agaagcgagt aggacccagc tttcttgtac aaagtggtcc cc   832 SEQ ID NO: 18 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg    60 (pLV430G human gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac   120 CD86 vector) cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat   180 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc   240 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac   300 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct   360 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc   420 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac   480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat   540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg   600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc   660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct   720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg   780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg   840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag   900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat   960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag  1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca  1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata  1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga  1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc  1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata  1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag  1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag  1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat  1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc  1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa  1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca  1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc  1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct  1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata  1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat  1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag  1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga  2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat  2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg  2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa  2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa  2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg  2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag  2400 agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag  2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc  2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca  2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc  2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat  2700 caacaagttt gtacaaaaaa gcaggcttcg ccaccatggg cctgagcaac atcctgttcg  2760 tgatggcctt cctgctgtcc ggagccgccc ctctgaagat ccaggcctac ttcaacgaga  2820 ccgccgacct gccctgccag ttcgccaaca gccagaacca gagcctgagc gaactggtgg  2880 tgttctggca ggaccaggaa aacctggtcc tgaacgaggt gtacctgggc aaagaaaagt  2940 tcgacagcgt gcacagcaag tacatgggcc ggaccagctt cgacagcgac agctggaccc  3000 tgcggctgca caacctgcag atcaaggaca agggcctgta ccagtgcatc atccaccaca  3060 agaaacccac cggcatgatc agaatccacc agatgaacag cgagctgtcc gtgctggcca  3120 acttcagcca gcccgagatc gtgcccatca gcaacatcac cgagaacgtg tacatcaacc  3180 tgacctgcag cagcatccac ggctaccccg agcccaagaa aatgagcgtg ctgctgcgga  3240 ccaagaacag caccatcgag tacgacggcg tgatgcagaa aagccaggac aacgtgaccg  3300 agctgtacga cgtgagcatc agcctgagcg tgagcttccc cgacgtgacc agcaacatga  3360 ccatcttttg catcctggaa accgacaaga cccggctgct gtccagcccc ttcagcatcg  3420 agctggaaga tccccagccc cctcccgacc acatcccctg gatcaccgcc gtgctgccca  3480 ccgtgatcat ctgcgtgatg gtgttctgcc tgatcctgtg gaagtggaag aagaagaagc  3540 ggcctaggaa cagctacaag tgcggcacca acaccatgga acgggaggaa agcgagcaga  3600 ccaagaagcg ggagaagatc cacatccccg agcggagcga cgaggcccag cgggtgttca  3660 agagcagcaa gaccagcagc tgcgacaaga gcgacacctg cttctaggac ccagctttct  3720 tgtacaaagt ggtgattcga gttaattaag ctagcctagt gccatttgtt cagtggttcg  3780 tagggctttc ccccactgtt tggctttcag ttatatggat gatgtggtat tgggggccaa  3840 gtctgtacag catcttgagt ccctttttac cgctgttacc aattttcttt tgtctttggg  3900 tatacattta aaccctaaca aaacaaagag atggggttac tctctaaatt ttatgggtta  3960 tgtcattgga tgttatgggt ccttgccaca agaacacatc atacaaaaaa tcaaagaatg  4020 ttttagaaaa cttcctatta acaggcctat tgattggaaa gtatgtcaac gaattgtggg  4080 tcttttgggt tttgctgccc cttttacaca atgtggttat cctgcgttga tgcctttgta  4140 tgcatgtatt caatctaagc aggctttcac tttctcgcca acttacaagg cctttctgtg  4200 taaacaatac ctgaaccttt accccgttgc ccggcaacgg ccaggtctgt gccaagtgtt  4260 tgctgacgca acccccactg gctggggctt ggtcatgggc catcagcgca tgcgtggaac  4320 cttttcggct cctctgccga tccatactgc ggaactccta gccgcttgtt ttgctcgcag  4380 caggtctgga gcaaacatta tcgggactga taactctgtt gtcctatccc gcaaatatac  4440 atcgtttcca tggctgctag gctgtgctgc caactggatc ctgcgcggga cgtcctttgt  4500 ttacgtcccg tcggcgctga atcctgcgga cgacccttct cggggtcgct tgggactctc  4560 tcgtcccctt ctccgtctgc cgttccgacc gaccacgggg cgcacctctc tttacgcgga  4620 ctccccgtct gtgccttctc atctgccgga ccgtgtgcac ttcgcttcac ctctgcacgt  4680 cgcatggaga ccaccgtgaa cgcccaccaa atattgccca aggtcttaca taagaggact  4740 cttggactct cagcaatgtc aacgaccgac cttgaggcat acttcaaaga ctgtttgttt  4800 aaagactggg aggagttggg ggaggagatt aggttaaagg tctttgtact aggaggctgt  4860 aggcataaat tggtctgcgc accagcacca tggcgcaatc actagagcgg ggtaccttta  4920 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga  4980 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct  5040 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt  5100 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac  5160 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt  5220 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt  5280 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat  5340 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat  5400 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa  5460 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag  5520 aggccgaggc cggatccctt gagtggcttt catcctggag cagactttgc agtctgtgga  5580 ctgcaacaca acattgcctt tatgtgtaac tcttggctga agctcttaca ccaatgctgg  5640 gggacatgta cctcccaggg gcccaggaag actacgggag gctacaccaa cgtcaatcag  5700 aggggcctgt gtagctaccg ataagcggac cctcaagagg gcattagcaa tagtgtttat  5760 aaggccccct tgttaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg  5820 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc  5880 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac  5940 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt  6000 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag  6060 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg  6120 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa  6180 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc  6240 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag  6300 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa  6360 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc  6420 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg  6480 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa  6540 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa  6600 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg  6660 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag  6720 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg  6780 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt  6840 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt  6900 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac  6960 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag  7020 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg  7080 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca  7140 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga  7200 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca  7260 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc  7320 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca  7380 ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa  7440 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc  7500 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc  7560 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg  7620 cctttttacg gttcctggcc ttttgctggc ctttttgaag ctgtccctga tggtcgtcat  7680 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa  7740 gaatcataat ggggaaggcc atccagcctc gcgtcg  7776 SEQ ID NO: 19 atgggcctga gcaacatcct gttcgtgatg gccttcctgc tgtccggagc cgcccctctg    60 (CD86 CoOP) aagatccagg cctacttcaa cgagaccgcc gacctgccct gccagttcgc caacagccag   120 aaccagagcc tgagcgaact ggtggtgttc tggcaggacc aggaaaacct ggtcctgaac   180 gaggtgtacc tgggcaaaga aaagttcgac agcgtgcaca gcaagtacat gggccggacc   240 agcttcgaca gcgacagctg gaccctgcgg ctgcacaacc tgcagatcaa ggacaagggc   300 ctgtaccagt gcatcatcca ccacaagaaa cccaccggca tgatcagaat ccaccagatg   360 aacagcgagc tgtccgtgct ggccaacttc agccagcccg agatcgtgcc catcagcaac   420 atcaccgaga acgtgtacat caacctgacc tgcagcagca tccacggcta ccccgagccc   480 aagaaaatga gcgtgctgct gcggaccaag aacagcacca tcgagtacga cggcgtgatg   540 cagaaaagcc aggacaacgt gaccgagctg tacgacgtga gcatcagcct gagcgtgagc   600 ttccccgacg tgaccagcaa catgaccatc ttttgcatcc tggaaaccga caagacccgg   660 ctgctgtcca gccccttcag catcgagctg gaagatcccc agccccctcc cgaccacatc   720 ccctggatca ccgccgtgct gcccaccgtg atcatctgcg tgatggtgtt ctgcctgatc   780 ctgtggaagt ggaagaagaa gaagcggcct aggaacagct acaagtgcgg caccaacacc   840 atggaacggg aggaaagcga gcagaccaag aagcgggaga agatccacat ccccgagcgg   900 agcgacgagg cccagcgggt gttcaagagc agcaagacca gcagctgcga caagagcgac   960 acctgcttc   969 SEQ ID NO: 20 ggggacaagt ttgtacaaaa aagcaggctt cgccaccatg ggcctgagca acatcctgtt    60 (CD86 PCRP) cgtgatggcc ttcctgctgt ccggagccgc ccctctgaag atccaggcct acttcaacga   120 gaccgccgac ctgccctgcc agttcgccaa cagccagaac cagagcctga gcgaactggt   180 ggtgttctgg caggaccagg aaaacctggt cctgaacgag gtgtacctgg gcaaagaaaa   240 gttcgacagc gtgcacagca agtacatggg ccggaccagc ttcgacagcg acagctggac   300 cctgcggctg cacaacctgc agatcaagga caagggcctg taccagtgca tcatccacca   360 caagaaaccc accggcatga tcagaatcca ccagatgaac agcgagctgt ccgtgctggc   420 caacttcagc cagcccgaga tcgtgcccat cagcaacatc accgagaacg tgtacatcaa   480 cctgacctgc agcagcatcc acggctaccc cgagcccaag aaaatgagcg tgctgctgcg   540 gaccaagaac agcaccatcg agtacgacgg cgtgatgcag aaaagccagg acaacgtgac   600 cgagctgtac gacgtgagca tcagcctgag cgtgagcttc cccgacgtga ccagcaacat   660 gaccatcttt tgcatcctgg aaaccgacaa gacccggctg ctgtccagcc ccttcagcat   720 cgagctggaa gatccccagc cccctcccga ccacatcccc tggatcaccg ccgtgctgcc   780 caccgtgatc atctgcgtga tggtgttctg cctgatcctg tggaagtgga agaagaagaa   840 gcggcctagg aacagctaca agtgcggcac caacaccatg gaacgggagg aaagcgagca   900 gaccaagaag cgggagaaga tccacatccc cgagcggagc gacgaggccc agcgggtgtt   960 caagagcagc aagaccagca gctgcgacaa gagcgacacc tgcttctagg acccagcttt  1020 cttgtacaaa gtggtcccc  1039 SEQ ID NO: 21 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga    60 (pDONR221 CD86 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga   120 vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca   180 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc   240 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta   300 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc   360 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa   420 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg   480 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa   540 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac   600 ctgttcgttg caacacattg atgagcaatg cttttttata atgcacaagt ttgtacaaaa   660 aagcaggctt cgccaccatg ggcctgagca acatcctgtt cgtgatggcc ttcctgctgt   720 ccggagccgc ccctctgaag atccaggcct acttcaacga gaccgccgac ctgccctgcc   780 agttcgccaa cagccagaac cagagcctga gcgaactggt ggtgttctgg caggaccagg   840 aaaacctggt cctgaacgag gtgtacctgg gcaaagaaaa gttcgacagc gtgcacagca   900 agtacatggg ccggaccagc ttcgacagcg acagctggac cctgcggctg cacaacctgc   960 agatcaagga caagggcctg taccagtgca tcatccacca caagaaaccc accggcatga  1020 tcagaatcca ccagatgaac agcgagctgt ccgtgctggc caacttcagc cagcccgaga  1080 tcgtgcccat cagcaacatc accgagaacg tgtacatcaa cctgacctgc agcagcatcc  1140 acggctaccc cgagcccaag aaaatgagcg tgctgctgcg gaccaagaac agcaccatcg  1200 agtacgacgg cgtgatgcag aaaagccagg acaacgtgac cgagctgtac gacgtgagca  1260 tcagcctgag cgtgagcttc cccgacgtga ccagcaacat gaccatcttt tgcatcctgg  1320 aaaccgacaa gacccggctg ctgtccagcc ccttcagcat cgagctggaa gatccccagc  1380 cccctcccga ccacatcccc tggatcaccg ccgtgctgcc caccgtgatc atctgcgtga  1440 tggtgttctg cctgatcctg tggaagtgga agaagaagaa gcggcctagg aacagctaca  1500 agtgcggcac caacaccatg gaacgggagg aaagcgagca gaccaagaag cgggagaaga  1560 tccacatccc cgagcggagc gacgaggccc agcgggtgtt caagagcagc aagaccagca  1620 gctgcgacaa gagcgacacc tgcttctagg acccagcttt cttgtacaaa gtggtcatta  1680 taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa  1740 tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg  1800 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa  1860 taatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta  1920 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt  1980 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct  2040 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca  2100 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga  2160 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg  2220 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg  2280 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca  2340 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg  2400 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc  2460 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata  2520 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg  2580 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat  2640 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt  2700 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc  2760 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga  2820 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag  2880 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg  2940 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca  3000 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga  3060 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca  3120 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc  3180 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca  3240 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa  3300 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc  3360 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc  3420 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg  3480 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt  3526 SEQ ID NO: 22 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga    60 (pDONR221 4- taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga   120 1BBL vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca   180 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc   240 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta   300 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc   360 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa   420 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg   480 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa   540 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac   600 ctgttcgttg caacacattg atgagcaatg cttttttata atgcacaagt ttgtacaaaa   660 aagcaggctt cgccaccatg gaatacgcct ctgatgccag cctggacccc gaagctcctt   720 ggcctcctgc ccctagagcc agagcctgta gagtgctgcc ttgggctctg gtggctggcc   780 ttctccttct gctgctgctg gccgctgcct gcgctgtgtt tctggcttgt ccttgggccg   840 tgtcaggcgc cagagcttct cctggatctg ccgccagccc cagactgaga gagggacctg   900 agctgagccc cgatgatcct gccggactgc tggatctgag acagggcatg ttcgcccagc   960 tggtggccca gaacgtgctg ctgatcgatg gccccctgag ctggtacagc gatcctggac  1020 tggctggcgt gtcactgaca ggcggcctga gctacaaaga ggacaccaaa gaactggtgg  1080 tggccaaggc cggcgtgtac tacgtgttct ttcagctgga actgcggaga gtggtggccg  1140 gcgaaggatc cggctctgtg tctctggcac tgcatctgca gcccctgaga tctgctgcag  1200 gcgctgctgc actggccctg acagtggacc tgcctccagc ctctagcgag gccagaaact  1260 ccgcattcgg gtttcaaggc agactgctgc acctgtctgc cggccagaga ctgggagtgc  1320 atctgcacac agaggccaga gccagacacg cctggcagct gacacagggc gctacagtgc  1380 tgggcctgtt cagagtgacc cccgaaattc cagccggcct gcccagccct agaagcgagt  1440 aggacccagc tttcttgtac aaagtggtca ttataagaaa gcattgctta tcaatttgtt  1500 gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgatatcc  1560 cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc  1620 aaaatctctg atgttacatt gcacaagata aaataatatc atcatgaaca ataaaactgt  1680 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga  1740 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata  1800 atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt  1860 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac  1920 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg  1980 atgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag  2040 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc  2100 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg  2160 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg  2220 gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt  2280 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa  2340 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc  2400 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg  2460 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct  2520 aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact tgaggggagg  2580 gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag  2640 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct  2700 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac  2760 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc  2820 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg  2880 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt  2940 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt  3000 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc  3060 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca  3120 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata  3180 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg  3240 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct  3300 ggccttttgc tcacatgtt  3319 SEQ ID NO: 23 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg    60 (pLV430G gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac   120 vector) cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat   180 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc   240 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac   300 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct   360 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc   420 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac   480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat   540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg   600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc   660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct   720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg   780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg   840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag   900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat   960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag  1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca  1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata  1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga  1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc  1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata  1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag  1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag  1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat  1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc  1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa  1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca  1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc  1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct  1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata  1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat  1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag  1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga  2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat  2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg  2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa  2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa  2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg  2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag  2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag  2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc  2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca  2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc  2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat  2700 cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt  2760 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc  2820 actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat  2880 gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag  2940 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt  3000 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg  3060 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt  3120 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg  3180 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt  3240 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa  3300 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg  3360 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat  3420 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag  3480 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc  3540 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt  3600 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt  3660 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc  3720 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt  3780 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga  3840 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa  3900 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata  3960 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg  4020 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg  4080 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg  4140 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg  4200 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac  4260 acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta  4320 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct  4380 cgttcagctt tcttgtacaa agtggtgatt cgagttaatt aagctagcct agtgccattt  4440 gttcagtggt tcgtagggct ttcccccact gtttggcttt cagttatatg gatgatgtgg  4500 tattgggggc caagtctgta cagcatcttg agtccctttt taccgctgtt accaattttc  4560 ttttgtcttt gggtatacat ttaaacccta acaaaacaaa gagatggggt tactctctaa  4620 attttatggg ttatgtcatt ggatgttatg ggtccttgcc acaagaacac atcatacaaa  4680 aaatcaaaga atgttttaga aaacttccta ttaacaggcc tattgattgg aaagtatgtc  4740 aacgaattgt gggtcttttg ggttttgctg ccccttttac acaatgtggt tatcctgcgt  4800 tgatgccttt gtatgcatgt attcaatcta agcaggcttt cactttctcg ccaacttaca  4860 aggcctttct gtgtaaacaa tacctgaacc tttaccccgt tgcccggcaa cggccaggtc  4920 tgtgccaagt gtttgctgac gcaaccccca ctggctgggg cttggtcatg ggccatcagc  4980 gcatgcgtgg aaccttttcg gctcctctgc cgatccatac tgcggaactc ctagccgctt  5040 gttttgctcg cagcaggtct ggagcaaaca ttatcgggac tgataactct gttgtcctat  5100 cccgcaaata tacatcgttt ccatggctgc taggctgtgc tgccaactgg atcctgcgcg  5160 ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc ggacgaccct tctcggggtc  5220 gcttgggact ctctcgtccc cttctccgtc tgccgttccg accgaccacg gggcgcacct  5280 ctctttacgc ggactccccg tctgtgcctt ctcatctgcc ggaccgtgtg cacttcgctt  5340 cacctctgca cgtcgcatgg agaccaccgt gaacgcccac caaatattgc ccaaggtctt  5400 acataagagg actcttggac tctcagcaat gtcaacgacc gaccttgagg catacttcaa  5460 agactgtttg tttaaagact gggaggagtt gggggaggag attaggttaa aggtctttgt  5520 actaggaggc tgtaggcata aattggtctg cgcaccagca ccatggcgca atcactagag  5580 cggggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa  5640 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc  5700 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg  5760 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg  5820 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat  5880 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg  5940 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa  6000 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc  6060 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc  6120 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt  6180 tttatttatg cagaggccga ggccggatcc cttgagtggc tttcatcctg gagcagactt  6240 tgcagtctgt ggactgcaac acaacattgc ctttatgtgt aactcttggc tgaagctctt  6300 acaccaatgc tgggggacat gtacctccca ggggcccagg aagactacgg gaggctacac  6360 caacgtcaat cagaggggcc tgtgtagcta ccgataagcg gaccctcaag agggcattag  6420 caatagtgtt tataaggccc ccttgttaat tcttgaagac gaaagggcct cgtgatacgc  6480 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt  6540 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat  6600 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg  6660 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt  6720 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga  6780 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa  6840 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt  6900 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt  6960 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc  7020 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga  7080 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat  7140 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct  7200 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc  7260 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg  7320 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc  7380 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg  7440 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca  7500 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta  7560 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc  7620 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa  7680 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca  7740 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta  7800 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc  7860 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca  7920 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta  7980 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag  8040 cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag cgccacgctt  8100 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc  8160 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac  8220 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac  8280 gccagcaacg cggccttttt acggttcctg gccttttgct ggcctttttg aagctgtccc  8340 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg  8400 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcg  8449 SEQ ID NO: 24 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga    60 (pDONR221 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga   120 vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca   180 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc   240 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta   300 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc   360 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa   420 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg   480 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa   540 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac   600 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa   660 agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa   720 aaaacagact acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt   780 agatggtatt agtgacctgt agtcgaccga cagccttcca aatgttcttc gggtgatgct   840 gccaacttag tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca   900 gcctactcgc tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg   960 tgcgagcctc ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt  1020 catagtcctg aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta  1080 caagtcgttc ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt  1140 ctgtaatttc tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat  1200 attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca  1260 gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc  1320 cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc  1380 agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc  1440 tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc  1500 atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac  1560 ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc  1620 attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac  1680 tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg  1740 acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat  1800 acgcatactg ttatctggct tttagtaagc cggatccacg cggcgtttac gccccgccct  1860 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac  1920 agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat  1980 atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa  2040 aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt  2100 tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa  2160 actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat  2220 ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg  2280 ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat  2340 aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg  2400 tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc  2460 attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag  2520 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt  2580 gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg  2640 gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca  2700 caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc gactacaggt  2760 cactaatacc atctaagtag ttgattcata gtgactggat atgttgtgtt ttacagtatt  2820 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg  2880 tttctcgttc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt  2940 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata  3000 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt  3060 ctcaaaatct ctgatgttac attgcacaag ataaaataat atcatcatga acaataaaac  3120 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt  3180 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg  3240 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag  3300 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca  3360 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc  3420 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag  3480 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt  3540 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc  3600 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta  3660 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg  3720 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat  3780 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca  3840 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat  3900 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt  3960 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg  4020 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt  4080 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct  4140 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc  4200 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc  4260 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc  4320 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg  4380 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt  4440 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg  4500 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg  4560 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt  4620 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag  4680 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt  4740 gctggccttt tgctcacatg t  4761 SEQ ID NO: 25 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt    60 (psPAX2 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca   120 plasmid) gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg   180 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca   240 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt   300 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt   360 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca   420 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca   480 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga   540 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact   600 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga   660 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg   720 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc   780 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga   840 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat   900 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt   960 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt  1020 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggt  1080 cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc  1140 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc  1200 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg  1260 actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat  1320 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc  1380 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta  1440 ttagtcatcg ctattaccat gggtcgaggt gagccccacg ttctgcttca ctctccccat  1500 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc  1560 gatgggggcg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg  1620 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc  1680 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg  1740 gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc  1800 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg  1860 gctgtaatta gcgcttggtt taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc  1920 ttaaagggct ccgggagggc cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg  1980 tgtgtgtgcg tggggagcgc cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg  2040 ggcgcggcgc ggggctttgt gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg  2100 tgccccgcgg tgcggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg  2160 ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt aacccccccc tgcaccgccc  2220 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc  2280 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc  2340 gcctcgggcc ggggagggct cgggggaggg gcgcggcggc cccggagcgc cggcggctgt  2400 cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga  2460 cttcctttgt cccaaatctg gcggagccga aatctgggag gcgccgccgc accccctcta  2520 gcgggcgcgg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg  2580 tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgcc gcagggggac  2640 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc  2700 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac  2760 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgggccggc cgcgttgacg  2820 cgcacggcaa gaggcgaggg gcggcgactg gtgagagatg ggtgcgagag cgtcagtatt  2880 aagcggggga gaattagatc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa  2940 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc  3000 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct  3060 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt  3120 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca  3180 aaacaaaagt aagaaaaaag cacagcaagc agcagctgac acaggacaca gcaatcaggt  3240 cagccaaaat taccctatag tgcagaacat ccaggggcaa atggtacatc aggccatatc  3300 acctagaact ttaaatgcat gggtaaaagt agtagaagag aaggctttca gcccagaagt  3360 gatacccatg ttttcagcat tatcagaagg agccacccca caagatttaa acaccatgct  3420 aaacacagtg gggggacatc aagcagccat gcaaatgtta aaagagacca tcaatgagga  3480 agctgcagaa tgggatagag tgcatccagt gcatgcaggg cctattgcac caggccagat  3540 gagagaacca aggggaagtg acatagcagg aactactagt acccttcagg aacaaatagg  3600 atggatgaca cataatccac ctatcccagt aggagaaatc tataaaagat ggataatcct  3660 gggattaaat aaaatagtaa gaatgtatag ccctaccagc attctggaca taagacaagg  3720 accaaaggaa ccctttagag actatgtaga ccgattctat aaaactctaa gagccgagca  3780 agcttcacaa gaggtaaaaa attggatgac agaaaccttg ttggtccaaa atgcgaaccc  3840 agattgtaag actattttaa aagcattggg accaggagcg acactagaag aaatgatgac  3900 agcatgtcag ggagtggggg gacccggcca taaagcaaga gttttggctg aagcaatgag  3960 ccaagtaaca aatccagcta ccataatgat acagaaaggc aattttagga accaaagaaa  4020 gactgttaag tgtttcaatt gtggcaaaga agggcacata gccaaaaatt gcagggcccc  4080 taggaaaaag ggctgttgga aatgtggaaa ggaaggacac caaatgaaag attgtactga  4140 gagacaggct aattttttag ggaagatctg gccttcccac aagggaaggc cagggaattt  4200 tcttcagagc agaccagagc caacagcccc accagaagag agcttcaggt ttggggaaga  4260 gacaacaact ccctctcaga aggaggagcc gatagacaag gaactgtatc ctttagcttc  4320 cctcagatca ctctttggca gcgacccctc gtcacaataa agataggggg gcaattaaag  4380 gaagctctat tagatacagg agcagatgat acagtattag aagaaatgaa tttgccagga  4440 agatggaaac caaaaatgat agggggaatt ggaggtttta tcaaagtagg acagtatgat  4500 cagatactca tagaaatctg cggacataaa gctataggta cagtattagt aggacctaca  4560 cctgtcaaca taattggaag aaatctgttg actcagattg gctgcacttt aaattttccc  4620 attagtccta ttgagactgt accagtaaaa ttaaagccag gaatggatgg cccaaaagtt  4680 aaacaatggc cattgacaga agaaaaaata aaagcattag tagaaatttg tacagaaatg  4740 gaaaaggaag gaaaaatttc aaaaattggg cctgaaaatc catacaatac tccagtattt  4800 gccataaaga aaaaagacag tactaaatgg agaaaattag tagatttcag agaacttaat  4860 aagagaactc aagatttctg ggaagttcaa ttaggaatac cacatcctgc agggttaaaa  4920 cagaaaaaat cagtaacagt actggatgtg ggcgatgcat atttttcagt tcccttagat  4980 aaagacttca ggaagtatac tgcatttacc atacctagta taaacaatga gacaccaggg  5040 attagatatc agtacaatgt gcttccacag ggatggaaag gatcaccagc aatattccag  5100 tgtagcatga caaaaatctt agagcctttt agaaaacaaa atccagacat agtcatctat  5160 caatacatgg atgatttgta tgtaggatct gacttagaaa tagggcagca tagaacaaaa  5220 atagaggaac tgagacaaca tctgttgagg tggggattta ccacaccaga caaaaaacat  5280 cagaaagaac ctccattcct ttggatgggt tatgaactcc atcctgataa atggacagta  5340 cagcctatag tgctgccaga aaaggacagc tggactgtca atgacataca gaaattagtg  5400 ggaaaattga attgggcaag tcagatttat gcagggatta aagtaaggca attatgtaaa  5460 cttcttaggg gaaccaaagc actaacagaa gtagtaccac taacagaaga agcagagcta  5520 gaactggcag aaaacaggga gattctaaaa gaaccggtac atggagtgta ttatgaccca  5580 tcaaaagact taatagcaga aatacagaag caggggcaag gccaatggac atatcaaatt  5640 tatcaagagc catttaaaaa tctgaaaaca ggaaaatatg caagaatgaa gggtgcccac  5700 actaatgatg tgaaacaatt aacagaggca gtacaaaaaa tagccacaga aagcatagta  5760 atatggggaa agactcctaa atttaaatta cccatacaaa aggaaacatg ggaagcatgg  5820 tggacagagt attggcaagc cacctggatt cctgagtggg agtttgtcaa tacccctccc  5880 ttagtgaagt tatggtacca gttagagaaa gaacccataa taggagcaga aactttctat  5940 gtagatgggg cagccaatag ggaaactaaa ttaggaaaag caggatatgt aactgacaga  6000 ggaagacaaa aagttgtccc cctaacggac acaacaaatc agaagactga gttacaagca  6060 attcatctag ctttgcagga ttcgggatta gaagtaaaca tagtgacaga ctcacaatat  6120 gcattgggaa tcattcaagc acaaccagat aagagtgaat cagagttagt cagtcaaata  6180 atagagcagt taataaaaaa ggaaaaagtc tacctggcat gggtaccagc acacaaagga  6240 attggaggaa atgaacaagt agatgggttg gtcagtgctg gaatcaggaa agtactattt  6300 ttagatggaa tagataaggc ccaagaagaa catgagaaat atcacagtaa ttggagagca  6360 atggctagtg attttaacct accacctgta gtagcaaaag aaatagtagc cagctgtgat  6420 aaatgtcagc taaaagggga agccatgcat ggacaagtag actgtagccc aggaatatgg  6480 cagctagatt gtacacattt agaaggaaaa gttatcttgg tagcagttca tgtagccagt  6540 ggatatatag aagcagaagt aattccagca gagacagggc aagaaacagc atacttcctc  6600 ttaaaattag caggaagatg gccagtaaaa acagtacata cagacaatgg cagcaatttc  6660 accagtacta cagttaaggc cgcctgttgg tgggcgggga tcaagcagga atttggcatt  6720 ccctacaatc cccaaagtca aggagtaata gaatctatga ataaagaatt aaagaaaatt  6780 ataggacagg taagagatca ggctgaacat cttaagacag cagtacaaat ggcagtattc  6840 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta  6900 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa  6960 aattttcggg tttattacag ggacaggaga gatccagttt ggaaaggacc agcaaagctc  7020 ctctggaaag gtgaaggggc agtagtaata caagataata gtgacataaa agtagtgcca  7080 agaagaaaag caaagatcat cagggattat ggaaaacaga tggcaggtga tgattgtgtg  7140 gcaagtagac aggatgagga ttaacacatg gaattctgca acaactgctg tttatccatt  7200 tcagaattgg gtgtcgacat agcagaatag gcgttactcg acagaggaga gcaagaaatg  7260 gagccagtag atcctagact agagccctgg aagcatccag gaagtcagcc taaaactgct  7320 tgtaccaatt gctattgtaa aaagtgttgc tttcattgcc aagtttgttt catgacaaaa  7380 gccttaggca tctcctatgg caggaagaag cggagacagc gacgaagagc tcatcagaac  7440 agtcagactc atcaagcttc tctatcaaag cagtaagtag tacatgtaat gcaacctata  7500 atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata  7560 gtaatcatag aatataggaa aatggccgct gatcttcaga cctggaggag gagatatgag  7620 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt  7680 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg  7740 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac  7800 gctgacggta caggccagac aattattgtc tggtatagtg caggaggaga acaatttgct  7860 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct  7920 ccaagcaaga atcctagctg tggaaagata cctaaaggat caacagctcc tagggatttg  7980 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa  8040 taaatctctg gaacagatct ggaatcacac gacctggatg gagtgggaca gagaaattaa  8100 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa  8160 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac  8220 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag  8280 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc  8340 gtttcagacc cacctcccaa tcccgagggg acccgacagg cccgaaggaa tagaagaaga  8400 aggtggagag agagacagag acagatccat tcgattagtg aacggatcct tggcacttat  8460 ctgggacgat ctgcggagcc tgtgcctctt cagctaccac cgcttgagag acttactctt  8520 gattgtaacg aggattgtgg aacttctggg acgcaggggg tgggaagccc tcaaatattg  8580 gtggaatctc ctacaatatt ggagtcagga gctaaagaat agtgctgtta gcttgctcaa  8640 tgccacagcc atagcagtag ctgaggggac agatagggtt atagaagtag tacaaggagc  8700 ttgtagagct attcgccaca tacctagaag aataagacag ggcttggaaa ggattttgct  8760 ataagctcga aacaaccggt acctctagaa ctatagctag cagatctttt tccctctgcc  8820 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa  8880 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat  8940 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat  9000 gccatatgct ggctgccatg aacaaaggtg gctataaaga ggtcatcagt atatgaaaca  9060 gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt  9120 tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt  9180 actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt  9240 atgaagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg  9300 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta  9360 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg  9420 ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat  9480 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc  9540 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga  9600 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac  9660 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat  9720 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat  9780 catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta  9840 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc  9900 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg  9960 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 10020 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 10080 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 10140 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 10200 cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 10260 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 10320 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 10380 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 10440 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 10500 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 10560 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 10620 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 10680 ggattttggt catgagatta tca 10703 SEQ ID NO: 26 gtcgacggat cgggagatca attccggcac ctgtcctacg agttgcatga taaagaagac    60 (pCIGO-VSV.G agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt   120 plasmid) gaaggctctc aagggcatcg gtcgatgcag gaaaaggaca agcagcgaaa attcacgccc   180 ccttgggagg tggcggcata tgcaaaggat agcactccca ctctactact gggtatcata   240 tgctgactgt atatgcatga ggatagcata tgctacccgg atacagatta ggatagcata   300 tactacccag atatagatta ggatagcata tgctacccag atatagatta ggatagccta   360 tgctacccag atataaatta ggatagcata tactacccag atatagatta ggatagcata   420 tgctacccag atatagatta ggatagccta tgctacccag atatagatta ggatagcata   480 tgctacccag atatagatta ggatagcata tgctatccag atatttgggt agtatatgct   540 acccagatat aaattaggat agcatatact accctaatct ctattaggat agcatatgct   600 acccggatac agattaggat agcatatact acccagatat agattaggat agcatatgct   660 acccagatat agattaggat agcctatgct acccagatat aaattaggat agcatatact   720 acccagatat agattaggat agcatatgct acccagatat agattaggat agcctatgct   780 acccagatat agattaggat agcatatgct atccagatat ttgggtagta tatgctaccc   840 atggcaacat tagcccaccg tgctctcagc gacctcgtga atatgaggac caacaaccct   900 gtgcttggcg ctcaggcgca agtgtgtgta atttgtcctc cagatcgcag caatcgcgcc   960 cctatcttgg cccgcccacc tacttatgca ggtattcccc ggggtgccat tagtggtttt  1020 gtgggcaagt ggtttgaccg cagtggttag cggggttaca atcagccaag ttattacacc  1080 cttattttac agtccaaaac cgcagggcgg cgtgtggggg ctgacgcgtg cccccactcc  1140 acaatttcaa aaaaaagagt ggccacttgt ctttgtttat gggccccatt ggcgtggagc  1200 cccgtttaat tttcgggggt gttagagaca accagtggag tccgctgctg tcggcgtcca  1260 ctctctttcc ccttgttaca aatagagtgt aacaacatgg ttcacctgtc ttggtccctg  1320 cctgggacac atcttaataa ccccagtatc atattgcact aggattatgt gttgcccata  1380 gccataaatt cgtgtgagat ggacatccag tctttacggc ttgtccccac cccatggatt  1440 tctattgtta aagatattca gaatgtttca ttcctacact agtatttatt gcccaagggg  1500 tttgtgaggg ttatattggt gtcatagcac aatgccacca ctgaaccccc cgtccaaatt  1560 ttattctggg ggcgtcacct gaaaccttgt tttcgagcac ctcacataca ccttactgtt  1620 cacaactcag cagttattct attagctaaa cgaaggagaa tgaagaagca ggcgaagatt  1680 caggagagtt cactgcccgc tccttgatct tcagccactg cccttgtgac taaaatggtt  1740 cactaccctc gtggaatcct gaccccatgt aaataaaacc gtgacagctc atggggtggg  1800 agatatcgct gttccttagg acccttttac taaccctaat tcgatagcat atgcttcccg  1860 ttgggtaaca tatgctattg aattagggtt agtctggata gtatatacta ctacccggga  1920 agcatatgct acccgtttag ggttaacaag ggggccttat aaacactatt gctaatgccc  1980 tcttgagggt ccgcttatcg gtagctacac aggcccctct gattgacgtt ggtgtagcct  2040 cccgtagtct tcctgggccc ctgggaggta catgtccccc agcattggtg taagagcttc  2100 agccaagagt tacacataaa ggcaatgttg tgttgcagtc cacagactgc aaagtctgct  2160 ccaggatgaa agccactcaa gggatcttca atattggcca ttagccatat tattcattgg  2220 ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa  2280 tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac  2340 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg  2400 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt  2460 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca  2520 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc  2580 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta  2640 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac  2700 catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg  2760 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg  2820 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt  2880 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatca ctagaagctt  2940 tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgcttctg acacaacagt  3000 ctcgaactta agctgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac  3060 aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg  3120 tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt  3180 ccactcccag ttcaattaca gctcttaagg ctagagtact taatacgact cactataggc  3240 tagcggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcaacagag  3300 atcgatctgt ttccttgaca ctatgaagtg ccttttgtac ttagcctttt tattcattgg  3360 ggtgaattgc aagttcacca tagtttttcc acacaaccaa aaaggaaact ggaaaaatgt  3420 tccttctaat taccattatt gcccgtcaag ctcagattta aattggcata atgacttaat  3480 aggcacagcc atacaagtca aaatgcccaa gagtcacaag gctattcaag cagacggttg  3540 gatgtgtcat gcttccaaat gggtcactac ttgtgatttc cgctggtatg gaccgaagta  3600 tataacacag tccatccgat ccttcactcc atctgtagaa caatgcaagg aaagcattga  3660 acaaacgaaa caaggaactt ggctgaatcc aggcttccct cctcaaagtt gtggatatgc  3720 aactgtgacg gatgccgaag cagtgattgt ccaggtgact cctcaccatg tgctggttga  3780 tgaatacaca ggagaatggg ttgattcaca gttcatcaac ggaaaatgca gcaattacat  3840 atgccccact gtccataact ctacaacctg gcattctgac tataaggtca aagggctatg  3900 tgattctaac ctcatttcca tggacatcac cttcttctca gaggacggag agctatcatc  3960 cctgggaaag gagggcacag ggttcagaag taactacttt gcttatgaaa ctggaggcaa  4020 ggcctgcaaa atgcaatact gcaagcattg gggagtcaga ctcccatcag gtgtctggtt  4080 cgagatggct gataaggatc tctttgctgc agccagattc cctgaatgcc cagaagggtc  4140 aagtatctct gctccatctc agacctcagt ggatgtaagt ctaattcagg acgttgagag  4200 gatcttggat tattccctct gccaagaaac ctggagcaaa atcagagcgg gtcttccaat  4260 ctctccagtg gatctcagct atcttgctcc taaaaaccca ggaaccggtc ctgctttcac  4320 cataatcaat ggtaccctaa aatactttga gaccagatac atcagagtcg atattgctgc  4380 tccaatcctc tcaagaatgg tcggaatgat cagtggaact accacagaaa gggaactgtg  4440 ggatgactgg gcaccatatg aagacgtgga aattggaccc aatggagttc tgaggaccag  4500 ttcaggatat aagtttcctt tatacatgat tggacatggt atgttggact ccgatcttca  4560 tcttagctca aaggctcagg tgttcgaaca tcctcacatt caagacgctg cttcgcaact  4620 tcctgatgat gagagtttat tttttggtga tactgggcta tccaaaaatc caatcgagct  4680 tgtagaaggt tggttcagta gttggaaaag ctctattgcc tcttttttct ttatcatagg  4740 gttaatcatt ggactattct tggttctccg agttggtatc catctttgca ttaaattaaa  4800 gcacaccaag aaaagacaga tttatacaga catagagatg aaccgacttg gaaagtaact  4860 caaatcctgc acaacagatt cttcatgttt ggaccaaatc aacttgtgat accatgctca  4920 aagaggcctc aattatattt gagtttttaa tttttatgga attctgcaga tatccatcac  4980 actggcggcc gctcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct  5040 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc  5100 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat  5160 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg  5220 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc  5280 tctatggctt ctgaggcgga aagaaccagc tgcattaatg aatcggccaa cgcgcgggga  5340 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg  5400 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag  5460 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc  5520 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca  5580 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt  5640 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc  5700 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc  5760 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc  5820 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact  5880 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg  5940 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta  6000 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca  6060 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa  6120 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg  6180 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc  6240 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg  6300 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat  6360 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg  6420 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa  6480 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca  6540 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc  6600 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt  6660 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa  6720 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat  6780 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct  6840 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga  6900 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag  6960 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga  7020 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca  7080 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg  7140 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc  7200 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag  7260 gggttccgcg cacatttccc cgaaaagtgc cacctgac  7298

Expression of CD86 and 4-1BBL on engineered MOLM-14 aAPCs (also referred to herein as aMOLM14 aAPCs) was confirmed using flow cytometry (Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA), with results shown in FIG. 12. aMOLM-14 aAPCs were γ-irradiated at 100 Gy and frozen.

Example 4—Expansion of Tumor Infiltrating Lymphocytes Using MOLM-14 Artificial Antigen Presenting Cells

Engineered MOLM-14 cells were gamma-irradiated at 100 Gy before co-culturing with TILs. REPs were initiated by culturing TILs with irradiated, engineered MOLM-14 cells at 1:100 ratios in CM2 media containing OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) for 14 days. At REP harvest, the TIL expansion rates, phenotype for activation and differentiation stage markers, metabolism rate, cytotoxicity and re-rapid expansion protocol (re-REP) assay were measured.

The results are shown in FIG. 13, FIG. 14, FIG. 15, and FIG. 16, where two expansions for two sets of patient TILs are compared. The results with the CD86/4-1BBL modified MOLM-14 cells (labeled “TIL+Engineered MOLM14+OKT3”) are comparable to the PBMC feeders (labeled “TIL+Feeders+OKT3”).

The results at day 14 are compared in FIG. 17, where results from two additional patient TILs are shown. The results indicate that MOLM-14 cells that were engineered with CD86 and 4-1BBL showed similar TIL expansion in the rapid expansion protocol when compared with allogeneic feeder cells. However, TILs cultured with parental MOLM-14 did not expand.

In addition, TILs expanded against MOLM-14 maintained a TIL phenotype and showed potency to kill P815 cells as measured using BRLA, which is described in detail in Example 9. Briefly, luciferin-transduced P815 target cells and TILs of interest were co-cultured with and without anti-CD3 to determine whether tumor reactivity of TILs is through TCR activation (specific killing) or non-specific killing. Following 4 hours of incubation, luciferin was added to the wells and incubated for 5 minutes. After the incubation, bioluminescence intensity was read using a luminometer. The percentage cytotoxicity and percentage survival were calculated using the following formula: % Survival=(experimental survival-minimum)/(maximum signal-minimum signal)×100 or % Cytotoxicity=100−(% Survival).

In FIG. 18, the results of expansions performed with low ratios of TILs to MOLM-14 aAPCs are shown in comparison to the results of expansions with PBMC feeders. TILs (2×10⁴) were cultured at different TIL to aAPC or PBMC ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”, respectively) with parental MOLM-14 (“MOLM14”) cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in a 24-well G-Rex plate. A control was performed using only OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) (“PBMC−”). Each condition was cultured in triplicate. Cultures were fed with fresh media and IL-2 on Day 4 and 7. Viable cells were counted on Day 7. FIG. 18 shows the mean plus standard deviation (SD) of viable cell numbers counted on Day 11, with a p-value calculated by the student t-test. Additional control experiments were performed using TILs alone, PBMCs alone, and aMOLM-14 cells alone, all of which resulted in undetectable cell numbers (data not shown). The results show that a ratio of 1:100 (TIL:aMOLM14) with OKT-3 and IL-2 yields a similar expansion when compared to PBMC feeders with OKT-3 and IL-2 (p=0.0598).

In FIG. 19, the results of expansions performed with higher ratios of TILs to MOLM-14 aAPCs, and otherwise performed as described above for FIG. 18, are shown in comparison to the results of expansions with PBMC feeders. At a ratio of 1:300, the CD86/4-1BBL modified MOLM-14 aAPCs with OKT-3 and IL-2 significantly outperform PBMC feeders with OKT-3 and IL-2. These results were verified using different TIL batches in repeat experiments shown in FIG. 20 and FIG. 21. In particular, as seen in FIG. 21, TIL to aMOLM14 ratios of 1:200 show enhanced TIL expansion compared to PBMC feeders under the same conditions. These results confirm that aMOLM14 aAPCs are unexpectedly superior in terms of expanding the TIL numbers than PBMCs particularly when using TIL:aMOLM14 ratios of 1:200 to 1:300.

In FIG. 22 and FIG. 23, TILs expanded with aMOLM14 or PBMC were compared by flow cytometry analysis to confirm that the TILs exhibited a similar phenotype and would be expected to perform similarly upon reinfusion into a patient. Briefly, TILs were first stained with L/D Aqua to determine viability. Next, cells were surface stained with TCR α/β PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28PE, CD27 APC-C7, and CD57-PerCP-Cy5.5. Phenotype analysis was done by gating 10,000 to 100,000 cells according to forward light scattering (FSC)/side light scattering (SSC) using a Canto II flow cytometer (Becton, Dickinson, and Co., Franklin Lakes, N.J., USA). Data was analyzed by Cytobank software to create sunburst diagrams and SPADE (Spanning Tree Progression of Density Normalized Event) analyses. Gates were set based on fluorescence minus one (FMO) controls. TILs expanded against aMOLM14 increases CD8⁺ TILs when compared to PBMC feeders. Without being bound by theory, this enhanced CD8⁺ TIL percentage may be due to the presence of 4-1BBL engineered to MOLM14. There is no difference in the expression of CD28, CD57, and CD27 differentiation markers. Additional flow cytometry data is shown in FIG. 24, and depicts a flow cytometry contour plot showing a memory subset (CD45RA+/−, CCR7+/−) gated on Live, TCR α/β+, CD4⁺ or CD8⁺ TILs, indicating that the memory subset obtained with PBMC feeders is replicated by the aMOLM14 aAPCs.

The CD4 and CD8 SPADE tree of TILs expanded with aMOLM14 aAPCs or PBMC feeders using CD3+ cells is shown in FIG. 25 and FIG. 26. The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT. Without being bound by theory, the results show that two batches of TILs expanded against aMOLM14 had undergone activation, but there was no difference in MFI between the aMOLM14 aAPCs and PBMC feeders, indicating that the aMOLM14 aAPCs effectively replicate the TIL phenotypic results obtained with PBMC feeders.

TILs expanded against aMOLM14 or PBMC were also analyzed for metabolic profiles. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TILs after expansion with irradiated PBMC feeders or aMOLM14 aAPCs were measured using a dual mitochondrial-glycolytic stress test. Briefly, cells were washed in assay medium (XF Assay Medium, Agilent Technologies, Santa Clara, Calif., USA), supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, at pH 7.4, and then 1×10⁵ viable cells were plated onto an adhesive-coated (Cell-Tak™, Corning) XFp cell culture microplate. Plates were spun to adhere the cells to the plate, then equilibrated at 37° C. in a humidified, non-CO₂ incubator prior to analysis of cellular metabolism. Mitochondrial and glycolytic stress test experiments were performed using a Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, Calif., USA), sequentially injecting the following compounds at specified intervals for simultaneous analysis of mitochondrial and glycolytic respiration of the cells: 1 μM oligomycin; 0.5 μM FCCP; 50 mM 2-deoxyglucose; and 0.5 μM each of rotenone and antimycin A. Results were analyzed using WAVE v2.3.0 software (Agilent Technologies, Santa Clara, Calif., USA) and GraphPad Prism v6.07 graphing software and are shown in FIG. 27 and FIG. 28, where points represent mean±SEM measured in triplicate. Both TILs grown with aMOLM14 aAPCs and PBMC feeders show similar oxphos and glycolysis behavior. This data suggests that aMOLM14 does not alter the metabolic programming of TILs when compared with PBMC feeders.

Example 5—Preparation of EM-3 Artificial Antigen Presenting Cells (aEM3 aAPCs)

EM-3 cells were obtained from Creative Bioarray, Inc. (Shirley, N.Y., USA). To develop an EM-3 based artificial APC, EM-3 cell lines were engineered with CD86, 4-1BBL, and antibody against IgG Fc region (Clone 7C12 or Clone 8B3). Human CD86 and human 4-1BBL/CD137 genes were cloned into commercially-available PLV430G and co-transfected with PDONR221 vectors (Invitrogen) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4, 571-589, to clone hCD86 and hCD137L genes onto the PLV430G and PDONR221 vectors. The 293T cell line was used for lentiviral production, and transduced to EM-3 cell lines. The transfected cells were sorted (S3e Cell Sorter, BioRad, Hercules, Calif., USA) using APC-conjugated CD86 and PE-conjugated CD137L to isolate and enrich the cells. The enriched cells were checked for purity by flow cytometry. Single-chain Fv (scFv) antibody clones designated 7C12 and 8B3 were generated against Fc of mouse IgG1, IgG2a and IgG2b (Viva Biotech Ltd., Chicago, Ill., USA). The amino acid sequences of these scFv clones are given in Table 7 (SEQ ID NO:27 and SEQ ID NO:28). The generated scFv clones were screened for Fc binding efficiency against OKT-3, engineered towards pLV4301G containing eGFP as co-reporter to produce lentivirus. The 293T cell line was used for packaging and lentiviral production. Engineered EM-3 (CD86/CD137L) cells were transduced using the lentiviral system and sorted using eGFP. EM37C12CD86CD137L and EM38B3CD86CD137L were regularly assessed for the consistent expression of each transduced molecule by flow cytometry.

TABLE 7 Amino acid sequences of scFv clones 7C12 and 8B3. Identifier (Description) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 27 QVQLVQSGGG LVKPGGSLRL SCAASGFNFN DQYMSWIRQA PGKGLEWVSF ISGSGGTTYY  60 (mFC-7C12 TDSVKGRFTI SRDNTKDSLY LQMNSLTVED TAVYYCARGG NYYTSVGRGT LVTVSAGGGG 120 scFv) SGAPDIQMTQ SPGTLSLSPG ERAILSCRAS QSVSGYLAWY QQKPGQAPRL LIYGASSRAT 180 GIPDRFSGSG SGTDFTLTIS SLRPEDIGTY YCKQYINAPF TFGGGTKVEI K 231 SEQ ID NO: 28 QVQLQQSGAE VKKPGSSVKV SCHASGGTFS SYAISWVRQA PGQGLEWMGW ISPYNGNTDY  60 (mFC-8B3 scFv) AQKVQGRVTL TTDTSTSTAY MELRSLRSDD TAVYYCATGG GTWYSDLWGR GTLVTVSAGG 120 GGSGGGGSGG GGSGAPEIVL TQSPSTLSAS VGDRVSITCR ASQSIGGSLA WYQQKPGKAP 180 KLLISEASTL ERGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQKYNSV PLTFGPGTKV 240 EIK 243

A non-limiting protocol for preparation of aEM3 aAPCs, which may also be adapted for use with aMOLM14 aAPCs, is described in the following paragraphs.

Molecular cloning of plasmids of interest may be performed as follows. To generate DONR vector the following cocktail may be used: B site flanked PCR product or destination vector (e.g., Gateway-adapted lentivector) 50-100 μg; DONR vector (e.g., pDONR222) 50-100 μg; BR Clonase II (Life Technologies) 1 μL; and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 μL). Incubate at room temperature for at least 1 hour. After incubation perform bacterial transformation either by heat shock method or electroporation. To generate destination vector, the following cocktail may be used: recombined pDONR vector (e.g., pDON222-geneX) 50-100 μg, destination vector (e.g., Gateway adapted lentivector) 50-100 μg, LR Clonase II (Life Technologies) 1 μL, and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 μL). Incubate at room temperature for at least 1 hour. After incubation, perform bacterial transformation either by chemical competent transformation/heat shock method.

Transformation and selection of the cloned plasmid may be performed as follows. The chemical competent transformation method may be performed as follows. Prepare nutrient agar plates (LB-Lennox or YT) with antibiotic for selection. Ensure that Recovery Medium (supplied by Lucigen, Middleton, Wis., USA) is readily available at room temperature. Optionally, sterile culture tubes may be chilled on ice (e.g., 17 mm×100 mm tubes (14 mL tube)), one tube for each transformation reaction). Remove E. cloni cells (Lucigen) from an −80° C. freezer and thaw completely on wet ice (5-15 minutes). Optionally add 40 μL of E. cloni cells to the chilled culture tube. Add 1-4 μL of DNA sample to the 40 μL of cells. Flick with finger (do not pipet up and down to mix, which can introduce air bubbles and warm the cells). Incubate the cell/DNA mixture on ice for 30 minutes. Heat shock cells by placing the culture tubes in a 42° C. water bath for 45 seconds. Return the 1.7 mL tube or culture tubes to ice for 2 minutes. Add 350 μL room temperature Recovery Medium to the cells or 960 μL of room temperature Recovery Medium to the cells in the culture tube. Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37° C. Plate up to 100% of the transformation mixture on LB-Lennox or YT agar plates containing the appropriate antibiotic. The plating volume may need to be optimized depending on DNA. Incubate the plates overnight at 37° C. Transformed clones can be further grown in any rich culture medium (e.g., LB or TB).

Colonies for Miniprep (Qiagen, Inc., Valencia, Calif., USA) may be grown as follows. After colonies have formed from plating recovered transformation reaction of DNA manipulation (e.g. LR reaction), add 1 mL desired TB/antibiotics into desired number of 2 mL Eppendorf microtubes with punctured caps. Pick desired number of colonies using ART LTS 20 μL soft pipette tip (VWR 89031-352) or 10 μL Denville tip. Place tip in 2 mL Eppendorf microtube with punctured cap. Cut the tip so that it fits in tube, close cap, and place tubes on shaker (purple 15 mL tube holder with VWR brand 15 mL tubes). Shake overnight (for no more than 16 hours) at 225 rpm/37° C. After overnight incubation, place each tip in a 1 mL tube in a ClavePak 96 plate from Denville with sterile water in it (to save the tip for making bacterial stock production after the plasmids are screened and selected). Perform Miniprep according to the Qiagen Mini prep kit protocol (Qiagen, Inc., Valencia, Calif., USA). Once the plasmids are eluted, restriction digestion is performed to select the right clones. After selecting the plasmids, use the tips saved from the same plasmids clone to grow the E. coli with the plasmid to make bacterial stock.

Lentiviral production may be performed as follows. The following media composition is prepared: 500 mL DMEM/F12 (Sigma); 25 mL FBS Heat Inactivated (HI) (Hyclone); 10 mM HEPES (Life Technologies); 1× Primocin (Invivogen); 1× Plasmocin (Invivogen); and 1× 2-mermactoethanol (Life Technologies). Harvest T75 flasks (Thermo Fisher Scientific) containing 90% confluent 293T cells. Aspirate media. Add 10 ml PBS, rinse gently and aspirate off. Add 2 mL TrypLE Express (Life Technologies) and evenly distribute it over the cell layer, let sit for 3-5 minutes at 37° C. (cell culture incubator). Add 10 mL media and disperse cells by pipetting up and down. Combine if there are multiple flasks. Count cells. If using a hemacytometer to determine concentration, cells/mL=(# counted cells×dilution factor×10⁴). To split back into T75 flasks, determine the time at which the cells will need to be fully confluent and dilute accordingly. (Cells double every 16-18 hours, so 3 days=1/27 dilution). Generally, a multiplication factor of 2.5 per day may be used where confluence is 2×10⁵ cells/cm². Bring volume up to 25 mL of media. To plate for titration of stocks, each well of the assay requires 5×10⁴ cells in 0.4 mL of media. Adjust 293T cells to 2×10⁴/mL in media. Plate 1 mL per well in a 24 well plate. For example, cells plated Monday may be infected on Tuesday and run on the flow cytometer on Friday, and cells plated Thursday are infected Friday and run on the flow cytometer on Monday. To plate for packaging transfections, seed T75 flasks with 6.8×10⁶ cells one day before transfection or 1.7×10⁶ cells on the morning of transfection. (Seeding on the day of transfection may reduce the variation in transfection efficiency). Bring volume in flask up to 25 mL with media. For example, flasks set up Monday are transfected Tuesday, and virus is collected on Thursday and Friday. In some cases (e.g., high titering constructs), the second collection can be omitted. To package lentiviral vectors, each T75 flask transfection requires 2 μg Baculo p35 plasmid (optional; only necessary if packaging a death gene), 2 μg VSV.G env plasmid (e.g., pMD2.G or PCIGO VSV-G); 4.7 μg Gag/polymerase plasmid (e.g., psPAX2 or pCMV-deltaR8.91), and 2.3 μg of the lentiviral vector described above. Determine the amount of VSV and R8.2/9.1 (+/−Baculo) plasmids needed for all samples (make a mixture of these DNAs if preparing many samples). Each T75 transfection requires 90 μL LipofectAmine 2000 (Thermo Fisher Scientific) in 2 mL Opti-MEM medium (Thermo Fisher Scientific). Make a mix containing enough Opti-Mem and LipofectAmine 2000 for all samples. Mix gently and let sit for 5 minutes at room temp, and label as tube A. For each transfection, add packaging DNA and specific lentiviral vector DNA to 500 μL room temperature Opti-MEM medium to a microtube and mix, and label as tube B. Add the 500 μL of DNA from tube B to the 2 mL of the LipofectAmine 2000 mix in tube A and mix gently, and incubate for 20-30 minutes at room temperature. Aspirate media from packaging flasks. Add the 2.5 mL of DNA/Lipofectamine complexes to 5 mL Opti-MEM medium and add to cells (do not pipet directly on cells since 293T cells are only semi adherent). Process plates in small groups to avoid drying. Incubate overnight and change media the next day in the morning. Collect the supernatant after 24 hours of media change. Supernatants can be harvested in a single collection, 48 hours after transfection or as 2 collections, 48 and 72 hours after transfection (in which case, harvests are pooled). If double collection is desired, collect supernatants by pipet on the first day, and replace with 20 mL of fresh media. To avoid flasks drying, work with only 5 flasks at a time. Keep collected supernatants at 4° C. until pooling the next day. Cool supernatants again on the following day and pool as appropriate. Spin the supernatants at 2000 rpm for 5 minutes to sediment any contaminating 293T cells. Filter harvested supernatants through a 0.45 μm or 0.8 μm filter unit containing a pre-filter disc. Use a large enough filtration unit so that the filtration speed is relatively fast. Store at 4° C. until ready to concentrate.

Virus may be concentrated using the PEG-it method (System Biosciences, Inc., Palo Alto, Calif. 94303) for longer-term storage at −80° C. Collect the supernatant from the transfection plates. Spin down the cell debris in the supernatant. The supernatant may also be filtered to completely remove any packaging cells. Add an amount of PEG-it solution equal to a quarter of the volume of supernatant to the supernatant. Incubate the suspension at 4° C. for overnight. Centrifuge at 3500 rpm (1500 g) at 4° C. for 30 minutes. Remove supernatant and centrifuge at 3500 rpm at 4° C. for 5 minutes. Remove remaining supernatant. Resuspend virus in desired amount of phosphate-buffered saline (PBS) and freeze aliquots at −80° C.

Transduction of cell line using lentivirus may be performed as follows. Adjust cells to be transduced to either: 1×10⁶ suspension cells per well in 24 well plate (1 well per transduction) or 50% confluence for adherent cells (1 well per transduction) in 24 well plate. For suspended cells, adjust concentration of cells to 1×10⁷/mL and plate 100 μL per well in 24 well plate (1 well per transduction). For adherent cells, plate to achieve 50% confluence on day of transduction based on cells/cm² (e.g., for 293T cells, confluence=2×10⁵/cm²). Total volume of transduction per well should be approximately 500 μL with 3-10 μg/mL Polybrene (Hexadimethrine bromide, Sigma-Aldrich Co., St. Louis, Miss., USA). The amount of concentrated virus added will depend on the MOI (multiplicity of infection) desired. A typical MOI is 10:1 but this may vary depending on cell type. The transfection well should contain 100 μL of standard media containing either 1×10⁶ suspension cells or 50% confluent cells. For a MOI of 10:1 (e.g., virus activity is 1×10⁸ IU/mL and the target is to infect 1×10⁶ cells, then 1×10⁷ virions or 100 μL of virus is needed). Add standard media to 500 μL. Add Polybrene to 3 μg/mL (primary cells) to 10 μg/mL (tumor cell lines). Spin plate(s) at 1800 rpm for 1.5 to 2 hours at 30° C. Incubate plate(s) at 37° C./5% CO₂ using a Tissue Culture incubator for 5 hours to overnight. Change media. After 72 hours of transduction, if enough cells are available, perform flow cytometric analysis to test the transduction efficiency.

Sorting of aAPCs may be performed as follows. Culture the cells in the media described above until the cell count reaches a minimum of 10-20 million. Take 1×10⁶ cells for each condition and stain with the antibodies for the proteins transduced. Wash the cells and analyze by flow cytometry to test the stability of transduction. Once the expression of protein of interest has been analyzed and confirmed, prepare the rest of the cells for sorting. Sort the cells in an S3 sorter by gating on markers of interest. Culture the sorted cells using the media mentioned above. Before freezing the vial, test the stability of the protein expression of interest. Use Recovery cell culture Freezing media (Invitrogen), to make the cell bank of the same cells. Cells may be banked after each transduction and sorting procedure.

Nucleotide sequence information for the 7C12 and 8B3 scFv clones (SEQ ID NO:29 and SEQ ID NO:30) and their lentiviral vectors are given in Table 8. Sequences used for generation of the pLV4301G 7C12 scFv mIgG hCD8 flag vector are provided as SED IQ NO:31 to SEQ ID NO:34 and are depicted in FIG. 29 to FIG. 32. Sequences used for generation of the pLV4301G 8B3 scFv mIgG hCD8 flag vector are provided as SEQ ID NO:35 to SEQ ID NO:38 and are depicted in FIG. 33 to FIG. 36.

TABLE 8 Nucleotide sequences for preparation of lentivirus for transduction of aAPCs. Identifier (Description) Sequence SEQ ID NO: 29 caggtgcagc tggtgcagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc   60 (mFC-7C12 tcctgtgcag cctctggatt caatttcaat gaccagtaca tgagttggat ccgccaggct  120 scFv) ccagggaagg ggctggagtg ggtttcattc attagtggta gtggtggtac cacatactac  180 acagactctg tgaagggccg gttcaccatc tccagggaca acaccaagga ctcattgtat  240 ttgcaaatga acagcctgac agtcgaggac acggccgtgt actactgtgc gagaggaggg  300 aattattata cttcggtggg ccggggcacc ctggtcaccg tctcggccgg tggcggcgga  360 tctggcgcgc cagacatcca gatgacccag tctccaggca ccctgtcttt gtctccaggg  420 gaaagagcca tcctctcctg cagggccagt cagagtgtta gcggctacct agcctggtat  480 caacagaaac ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact  540 ggcatcccag acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc  600 agcctgcggc ctgaagatat tggaacatat tactgtaaac agtacattaa tgccccattc  660 actttcggcg gcgggaccaa ggtggagatc aaa  693 SEQ ID NO: 30 caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc   60 (mFC-8B3 scFv) tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc  120 cctggacaag ggcttgagtg gatgggatgg atcagccctt acaatggtaa cacagattat  180 gcacagaagg tccagggcag agtcaccttg accacagaca catccacgag cacagcctac  240 atggagctga ggagcctgag atccgacgac acggccgtgt attactgtgc gacaggtggc  300 gggacctggt actccgatct ctggggccgt ggcaccctgg tcaccgtctc ggccggtggc  360 ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgccaga aattgtgctg  420 actcagtctc cctccaccct gtctgcatct gtaggagaca gagtcagcat cacttgccgg  480 gccagtcaga gtattggtgg gtcgttggcc tggtatcaac aaaagccagg gaaagcccct  540 aagctcctga tctctgaggc gtctacttta gagaggggcg tcccatcaag attcagcggc  600 agtggatctg ggacagattt cactctcacc atcaggagcc tgcagcctga agatgttgca  660 acttattact gtcaaaaata taacagtgtc ccgctcactt tcggccctgg gaccaaggtg  720 gagatcaaa  729 SEQ ID NO: 31 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg   60 (destination gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac  120 vector cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat  180 pLV4301G) taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc  240 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac  300 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct  360 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc  420 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac  480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat  540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg  600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc  660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct  720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg  780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg  840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag  900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat  960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700 cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820 actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880 gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaatggat 3600 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3660 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3720 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3780 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3840 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3900 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 3960 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4020 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4080 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4140 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4200 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4260 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4320 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4380 cagctttctt gtacaaagtg gtgattcgag ttaattaagt taacgaattc cccccctctc 4440 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4500 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4560 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4620 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4680 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4740 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 4800 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 4860 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 4920 ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 4980 tttcctttga aaaacacgat gataatatgg ccacaaccat gggaggcgga agcggcggag 5040 gctcccctcg aggcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 5100 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 5160 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 5220 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 5280 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 5340 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 5400 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 5460 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 5520 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 5580 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 5640 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 5700 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 5760 acgagctgta caagtaacgc gtcccgggtc tagagctagc ggtaccatgc attacgtagt 5820 cgacgactta attaagctag cctagtgcca tttgttcagt ggttcgtagg gctttccccc 5880 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 5940 ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 6000 ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 6060 atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 6120 ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 6180 ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 6240 ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 6300 acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 6360 ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 6420 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 6480 acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 6540 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 6600 cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 6660 gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 6720 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 6780 cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 6840 aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 6900 gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 6960 ctgcgcacca gcaccatggc gcaatcacta gagcggggta cctttaagac caatgactta 7020 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 7080 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 7140 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 7200 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 7260 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 7320 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 7380 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 7440 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7500 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7560 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgga 7620 tcccttgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat 7680 tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc 7740 ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag 7800 ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt 7860 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 7920 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7980 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 8040 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 8100 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 8160 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 8220 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 8280 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 8340 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 8400 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 8460 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 8520 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 8580 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 8640 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 8700 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 8760 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 8820 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 8880 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 8940 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 9000 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 9060 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 9120 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 9180 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 9240 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 9300 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 9360 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 9420 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 9480 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 9540 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 9600 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 9660 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 9720 ctggcctttt gctggccttt ttgaagctgt ccctgatggt cgtcatctac ctgcctggac 9780 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg 9840 aaggccatcc agcctcgcgt cg 9862 SEQ ID NO: 32 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc   60 (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga  120 1, pMK 7c12 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt  180 anti mFC scFV gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt  240 CoOp ECORV gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg  300 SacII L1R5) acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca  360 aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg  420 atgagcaatg cttttttata atgccaactt tgtacaaaaa agctgaacga tatcgccacc  480 atgggcagca cagccattct ggccctgctg ctggcagtgc tgcagggcgt gtcagctcag  540 gtgcagctgg tgcagtctgg cggcggactc gtgaaacctg gcggcagcct gagactgagc  600 tgtgccgcca gcggcttcaa cttcaacgac cagtacatga gctggatccg gcaggcccct  660 ggcaagggac tggaatgggt gtccttcatc agcggcagcg gcggcaccac ctactacacc  720 gatagcgtga agggccggtt caccatcagc cgggacaaca ccaaggacag cctgtacctg  780 cagatgaaca gcctgaccgt ggaagatacc gccgtgtact actgcgccag aggcggcaat  840 tactacacca gcgtgggcag aggcaccctc gtgacagtgt ctgctggcgg aggcggatca  900 ggcggcggag gatcaggggg aggcggaagc ggagcacccg atatccagat gacacagagc  960 cccggcaccc tgtctctgag ccctggcgaa agagccatcc tgagctgcag agccagccag 1020 agcgtgtccg gatacctggc ttggtatcag cagaagcccg gccaggcccc cagactgctg 1080 atctatggcg ccaggaggag agccacaggc atccccgata gattcagcgg ctctggcagc 1140 ggcaccgact tcaccctgac aatcagctcc ctgcggcccg aggacatcgg cacctactat 1200 tgcaagcagt acatcaacgc ccccttcacc ttcggcggag gcaccaaggt ggaaatcaag 1260 ccgcgggcca actttgtata caaaagtgga acgagaaacg taaaatgata taaatatcaa 1320 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat 1380 ccagtcacta tgaatcaact acttagatgg tattagtgac ctgtactggg cctcatgggc 1440 cttcctttca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaacatgg 1500 tcatagctgt ttccttgcgt attgggcgct ctccgcttcc tcgctcactg actcgctgcg 1560 ctcggtcgtt cgggtaaagc ctggggtgcc taatgagcaa aaggccagca aaaggccagg 1620 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgccgccc tgacgagcat 1680 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 1740 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 1800 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 1860 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 1920 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 1980 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2040 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 2100 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2160 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 2220 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 2280 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 2340 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2400 tctgacagtt attagaaaaa ttcatccagc agacgataaa acgcaatacg ctggctatcc 2460 ggtgccgcaa tgccatacag caccagaaaa cgatccgccc attcgccgcc cagttcttcc 2520 gcaatatcac gggtggccag cgcaatatcc tgataacgat ccgccacgcc cagacggccg 2580 caatcaataa agccgctaaa acggccattt tccaccataa tgttcggcag gcacgcatca 2640 ccatgggtca ccaccagatc ttcgccatcc ggcatgctcg ctttcagacg cgcaaacagc 2700 tctgccggtg ccaggccctg atgttcttca tccagatcat cctgatccac caggcccgct 2760 tccatacggg tacgcgcacg ttcaatacga tgtttcgcct gatgatcaaa cggacaggtc 2820 gccgggtcca gggtatgcag acgacgcatg gcatccgcca taatgctcac tttttctgcc 2880 ggcgccagat ggctagacag cagatcctga cccggcactt cgcccagcag cagccaatca 2940 cggcccgctt cggtcaccac atccagcacc gccgcacacg gaacaccggt ggtggccagc 3000 cagctcagac gcgccgcttc atcctgcagc tcgttcagcg caccgctcag atcggttttc 3060 acaaacagca ccggacgacc ctgcgcgctc agacgaaaca ccgccgcatc agagcagcca 3120 atggtctgct gcgcccaatc atagccaaac agacgttcca cccacgctgc cgggctaccc 3180 gcatgcaggc catcctgttc aatcatactc ttcctttttc aatattattg aagcatttat 3240 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 3300 ggggttccgc gcacatttcc ccgaaaagtg ccac 3334 SEQ ID NO: 33 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc   60 (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga  120 2, pMK hCD8a gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt  180 scaffold TN L5 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt  240 L2) gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg  300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca  360 aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg  420 atgagcaatg cttttttata atgcccaact ttgtatacaa aagtggcccg cggacaacaa  480 cccctgcccc cagacctcct accccagccc ctacaattgc cagccagcct ctgagcctga  540 ggcccgaggc ttgtagacct gctgctggcg gagccgtgca caccagagga ctggatttcg  600 cctgcgacat ctacatctgg gcccctctgg ccggcacatg tggcgtgctg ctgctgagcc  660 tcgtgatcac cctgtactgc ggctccacca gcggctccgg caagcccggc tctggcgagg  720 gctccaccag cggcgactac aaggacgacg atgacaagta ataggatatc ggttcagctt  780 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg  840 tcactatcag tcaaaataaa atcattattt ctgggcctca tgggccttcc tttcactgcc  900 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct  960 tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt 1020 aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1080 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1140 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1200 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1260 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1320 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1380 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1440 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1500 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 1560 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1620 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1680 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1740 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1800 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag 1860 aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca 1920 tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg 1980 gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg 2040 ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc 2100 agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg 2160 ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc 2220 gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta 2280 tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta 2340 gacagcagat cctgacccgg cacttcgccc agcagcagcc aatcacggcc cgcttcggtc 2400 accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc 2460 gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga 2520 cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc 2580 caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc 2640 tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2700 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2760 tttccccgaa aagtgccac 2779 SEQ ID NO: 34 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg   60 (Final vector gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac  120 used for cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat  180 lentiviral taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc  240 production, ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac  300 pLV4301G 7C12 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct  360 scFV mIgG hCD8 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc  420 flag) accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac  480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat  540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg  600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc  660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct  720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg  780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg  840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag  900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat  960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700 caccaacttt gtacaaaaaa gctgaacgat atcgccacca tgggcagcac agccattctg 2760 gccctgctgc tggcagtgct gcagggcgtg tcagctcagg tgcagctggt gcagtctggc 2820 ggcggactcg tgaaacctgg cggcagcctg agactgagct gtgccgccag cggcttcaac 2880 ttcaacgacc agtacatgag ctggatccgg caggcccctg gcaagggact ggaatgggtg 2940 tccttcatca gcggcagcgg cggcaccacc tactacaccg atagcgtgaa gggccggttc 3000 accatcagcc gggacaacac caaggacagc ctgtacctgc agatgaacag cctgaccgtg 3060 gaagataccg ccgtgtacta ctgcgccaga ggcggcaatt actacaccag cgtgggcaga 3120 ggcaccctcg tgacagtgtc tgctggcgga ggcggatcag gcggcggagg atcaggggga 3180 ggcggaagcg gagcacccga tatccagatg acacagagcc ccggcaccct gtctctgagc 3240 cctggcgaaa gagccatcct gagctgcaga gccagccaga gcgtgtccgg atacctggct 3300 tggtatcagc agaagcccgg ccaggccccc agactgctga tctatggcgc caggaggaga 3360 gccacaggca tccccgatag attcagcggc tctggcagcg gcaccgactt caccctgaca 3420 atcagctccc tgcggcccga ggacatcggc acctactatt gcaagcagta catcaacgcc 3480 cccttcacct tcggcggagg caccaaggtg gaaatcaagc cgcgggccaa ctttgtatac 3540 aaaagtggcc cgcggacaac aacccctgcc cccagacctc ctaccccagc ccctacaatt 3600 gccagccagc ctctgagcct gaggcccgag gcttgtagac ctgctgctgg cggagccgtg 3660 cacaccagag gactggattt cgcctgcgac atctacatct gggcccctct ggccggcaca 3720 tgtggcgtgc tgctgctgag cctcgtgatc accctgtact gcggctccac cagcggctcc 3780 ggcaagcccg gctctggcga gggctccacc agcggcgact acaaggacga cgatgacaag 3840 taataggata tcggttcagc tttcttgtac aaagttggga ttcgagttaa ttaagttaac 3900 gaattccccc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa 3960 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg 4020 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc 4080 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct 4140 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac 4200 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc 4260 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta 4320 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg 4380 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta ggccccccga 4440 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggga 4500 ggcggaagcg gcggaggctc ccctcgaggc accatggtga gcaagggcga ggagctgttc 4560 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 4620 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 4680 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 4740 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 4800 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 4860 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 4920 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 4980 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 5040 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 5100 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 5160 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 5220 atcactctcg gcatggacga gctgtacaag taacgcgtcc cgggtctaga gctagcggta 5280 ccatgcatta cgtagtcgac gacttaatta agctagccta gtgccatttg ttcagtggtt 5340 cgtagggctt tcccccactg tttggctttc agttatatgg atgatgtggt attgggggcc 5400 aagtctgtac agcatcttga gtcccttttt accgctgtta ccaattttct tttgtctttg 5460 ggtatacatt taaaccctaa caaaacaaag agatggggtt actctctaaa ttttatgggt 5520 tatgtcattg gatgttatgg gtccttgcca caagaacaca tcatacaaaa aatcaaagaa 5580 tgttttagaa aacttcctat taacaggcct attgattgga aagtatgtca acgaattgtg 5640 ggtcttttgg gttttgctgc cccttttaca caatgtggtt atcctgcgtt gatgcctttg 5700 tatgcatgta ttcaatctaa gcaggctttc actttctcgc caacttacaa ggcctttctg 5760 tgtaaacaat acctgaacct ttaccccgtt gcccggcaac ggccaggtct gtgccaagtg 5820 tttgctgacg caacccccac tggctggggc ttggtcatgg gccatcagcg catgcgtgga 5880 accttttcgg ctcctctgcc gatccatact gcggaactcc tagccgcttg ttttgctcgc 5940 agcaggtctg gagcaaacat tatcgggact gataactctg ttgtcctatc ccgcaaatat 6000 acatcgtttc catggctgct aggctgtgct gccaactgga tcctgcgcgg gacgtccttt 6060 gtttacgtcc cgtcggcgct gaatcctgcg gacgaccctt ctcggggtcg cttgggactc 6120 tctcgtcccc ttctccgtct gccgttccga ccgaccacgg ggcgcacctc tctttacgcg 6180 gactccccgt ctgtgccttc tcatctgccg gaccgtgtgc acttcgcttc acctctgcac 6240 gtcgcatgga gaccaccgtg aacgcccacc aaatattgcc caaggtctta cataagagga 6300 ctcttggact ctcagcaatg tcaacgaccg accttgaggc atacttcaaa gactgtttgt 6360 ttaaagactg ggaggagttg ggggaggaga ttaggttaaa ggtctttgta ctaggaggct 6420 gtaggcataa attggtctgc gcaccagcac catggcgcaa tcactagagc ggggtacctt 6480 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 6540 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 6600 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 6660 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 6720 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 6780 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga 6840 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 6900 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 6960 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct 7020 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 7080 agaggccgag gccggatccc ttgagtggct ttcatcctgg agcagacttt gcagtctgtg 7140 gactgcaaca caacattgcc tttatgtgta actcttggct gaagctctta caccaatgct 7200 gggggacatg tacctcccag gggcccagga agactacggg aggctacacc aacgtcaatc 7260 agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc aatagtgttt 7320 ataaggcccc cttgttaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata 7380 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 7440 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 7500 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 7560 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 7620 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 7680 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 7740 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 7800 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 7860 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 7920 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 7980 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 8040 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc 8100 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 8160 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 8220 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 8280 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 8340 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 8400 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 8460 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 8520 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 8580 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 8640 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 8700 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 8760 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 8820 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 8880 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8940 caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag 9000 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 9060 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 9120 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 9180 ggccttttta cggttcctgg ccttttgctg gcctttttga agctgtccct gatggtcgtc 9240 atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag 9300 aagaatcata atggggaagg ccatccagcc tcgcgtcg 9338 SEQ ID NO: 35 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg   60 (destination gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac  120 vector, cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat  180 pLV4301G) taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc  240 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac  300 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct  360 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc  420 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac  480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat  540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg  600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc  660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct  720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg  780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg  840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag  900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat  960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700 cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820 actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880 gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaatggat 3600 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3660 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3720 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3780 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3840 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3900 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 3960 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4020 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4080 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4140 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4200 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4260 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4320 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4380 cagctttctt gtacaaagtg gtgattcgag ttaattaagt taacgaattc cccccctctc 4440 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4500 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4560 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4620 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4680 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4740 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 4800 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 4860 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 4920 ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 4980 tttcctttga aaaacacgat gataatatgg ccacaaccat gggaggcgga agcggcggag 5040 gctcccctcg aggcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 5100 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 5160 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 5220 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 5280 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 5340 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 5400 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 5460 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 5520 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 5580 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 5640 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 5700 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 5760 acgagctgta caagtaacgc gtcccgggtc tagagctagc ggtaccatgc attacgtagt 5820 cgacgactta attaagctag cctagtgcca tttgttcagt ggttcgtagg gctttccccc 5880 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 5940 ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 6000 ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 6060 atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 6120 ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 6180 ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 6240 ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 6300 acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 6360 ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 6420 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 6480 acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 6540 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 6600 cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 6660 gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 6720 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 6780 cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 6840 aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 6900 gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 6960 ctgcgcacca gcaccatggc gcaatcacta gagcggggta cctttaagac caatgactta 7020 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 7080 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 7140 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 7200 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 7260 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 7320 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 7380 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 7440 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7500 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7560 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgga 7620 tcccttgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat 7680 tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc 7740 ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag 7800 ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt 7860 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 7920 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7980 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 8040 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 8100 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 8160 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 8220 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 8280 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 8340 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 8400 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 8460 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 8520 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 8580 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 8640 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 8700 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 8760 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 8820 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 8880 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 8940 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 9000 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 9060 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 9120 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 9180 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 9240 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 9300 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 9360 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 9420 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 9480 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 9540 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 9600 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 9660 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 9720 ctggcctttt gctggccttt ttgaagctgt ccctgatggt cgtcatctac ctgcctggac 9780 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg 9840 aaggccatcc agcctcgcgt cg 9862 SEQ ID NO: 36 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc   60 (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga  120 1, pMK 8B3 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt  180 anti mFC scFV gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt  240 CoOp ECORV gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg  300 SacII L1R5) acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca  360 aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg  420 atgagcaatg cttttttata atgccaactt tgtacaaaaa agctgaacga tatcgccacc  480 atgggcagca cagccattct ggccctgctg ctggcagtgc tgcagggcgt gtcagctcag  540 gtgcagctgc agcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc  600 tgcaaggcta gcggcggcac cttcaggagc tacgccattt cttgggtgcg ccaggcccct  660 ggacagggcc tggaatggat gggctggatc agcccctaca acggcaacac cgactacgcc  720 cagaaagtgc agggcagagt gaccctgacc accgacacca gcacctccac cgcctacatg  780 gaactgcgga gcctgagaag cgacgacacc gccgtgtact actgtgccac aggcggcgga  840 acctggtaca gcgatctgtg gggcagaggc accctcgtga cagtgtctgc tggcggcgga  900 ggatctggcg gaggcggaag tggcggggga ggaagcggag cacctgagat cgtgctgacc  960 cagagcccta gcacactgag cgccagcgtg ggcgacagag tgtccatcac ctgtagagcc 1020 agccagagca tcggaggcag cctggcctgg tatcagcaga agcctggcaa ggcccccaag 1080 ctgctgatct ctgaggccag caccctggaa agaggcgtgc ccagcagatt ttccggcagc 1140 ggctctggca ccgacttcac cctgacaatc agcagcctgc agcccgagga cgtggccacc 1200 tactactgcc agaagtacaa cagcgtgccc ctgaccttcg gccctggcac caaggtggaa 1260 atcaagccgc gggccaactt tgtatacaaa agtggaacga gaaacgtaaa atgatataaa 1320 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca 1380 acatatccag tcactatgaa tcaactactt agatggtatt agtgacctgt actgggcctc 1440 atgggccttc ctttcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 1500 acatggtcat agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc 1560 gctgcgctcg gtcgttcggg taaagcctgg ggtgcctaat gagcaaaagg ccagcaaaag 1620 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 1680 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 1740 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 1800 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 1860 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1920 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 1980 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2040 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 2100 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2160 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2220 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2280 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 2340 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 2400 acttggtctg acagttatta gaaaaattca tccagcagac gataaaacgc aatacgctgg 2460 ctatccggtg ccgcaatgcc atacagcacc agaaaacgat ccgcccattc gccgcccagt 2520 tcttccgcaa tatcacgggt ggccagcgca atatcctgat aacgatccgc cacgcccaga 2580 cggccgcaat caataaagcc gctaaaacgg ccattttcca ccataatgtt cggcaggcac 2640 gcatcaccat gggtcaccac cagatcttcg ccatccggca tgctcgcttt cagacgcgca 2700 aacagctctg ccggtgccag gccctgatgt tcttcatcca gatcatcctg atccaccagg 2760 cccgcttcca tacgggtacg cgcacgttca atacgatgtt tcgcctgatg atcaaacgga 2820 caggtcgccg ggtccagggt atgcagacga cgcatggcat ccgccataat gctcactttt 2880 tctgccggcg ccagatggct agacagcaga tcctgacccg gcacttcgcc cagcagcagc 2940 caatcacggc ccgcttcggt caccacatcc agcaccgccg cacacggaac accggtggtg 3000 gccagccagc tcagacgcgc cgcttcatcc tgcagctcgt tcagcgcacc gctcagatcg 3060 gttttcacaa acagcaccgg acgaccctgc gcgctcagac gaaacaccgc cgcatcagag 3120 cagccaatgg tctgctgcgc ccaatcatag ccaaacagac gttccaccca cgctgccggg 3180 ctacccgcat gcaggccatc ctgttcaatc atactcttcc tttttcaata ttattgaagc 3240 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 3300 caaatagggg ttccgcgcac atttccccga aaagtgccac 3340 SEQ ID NO: 37 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc   60 (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga  120 2, pMK hCD8a gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt  180 scaffold TN L5 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt  240 L2) gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg  300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca  360 aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg  420 atgagcaatg cttttttata atgcccaact ttgtatacaa aagtggcccg cggacaacaa  480 cccctgcccc cagacctcct accccagccc ctacaattgc cagccagcct ctgagcctga  540 ggcccgaggc ttgtagacct gctgctggcg gagccgtgca caccagagga ctggatttcg  600 cctgcgacat ctacatctgg gcccctctgg ccggcacatg tggcgtgctg ctgctgagcc  660 tcgtgatcac cctgtactgc ggctccacca gcggctccgg caagcccggc tctggcgagg  720 gctccaccag cggcgactac aaggacgacg atgacaagta ataggatatc ggttcagctt  780 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg  840 tcactatcag tcaaaataaa atcattattt ctgggcctca tgggccttcc tttcactgcc  900 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct  960 tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt 1020 aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1080 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1140 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1200 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1260 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1320 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1380 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1440 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1500 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 1560 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1620 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1680 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1740 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1800 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag 1860 aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca 1920 tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg 1980 gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg 2040 ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc 2100 agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg 2160 ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc 2220 gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta 2280 tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta 2340 gacaggagat cctgacccgg cacttcgccc aggaggagcc aatcacggcc cgcttcggtc 2400 accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc 2460 gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga 2520 cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc 2580 caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc 2640 tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2700 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2760 tttccccgaa aagtgccac 2779 SEQ ID NO: 38 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg   60 (Final vector gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac  120 used for cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat  180 lentiviral taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc  240 production, ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac  300 pLV4301G 8B3 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct  360 scFV mIgG hCD8 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc  420 flag) accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac  480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat  540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg  600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc  660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct  720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg  780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg  840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag  900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat  960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700 caccaacttt gtacaaaaaa gctgaacgat atcgccacca tgggcagcac agccattctg 2760 gccctgctgc tggcagtgct gcagggcgtg tcagctcagg tgcagctgca gcagtctggc 2820 gccgaagtga agaaacccgg cagcagcgtg aaggtgtcct gcaaggctag cggcggcacc 2880 ttcaggagct acgccatttc ttgggtgcgc caggcccctg gacagggcct ggaatggatg 2940 ggctggatca gcccctacaa cggcaacacc gactacgccc agaaagtgca gggcagagtg 3000 accctgacca ccgacaccag cacctccacc gcctacatgg aactgcggag cctgagaagc 3060 gacgacaccg ccgtgtacta ctgtgccaca ggcggcggaa cctggtacag cgatctgtgg 3120 ggcagaggca ccctcgtgac agtgtctgct ggcggcggag gatctggcgg aggcggaagt 3180 ggcgggggag gaagcggagc acctgagatc gtgctgaccc agagccctag cacactgagc 3240 gccagcgtgg gcgacagagt gtccatcacc tgtagagcca gccagagcat cggaggcagc 3300 ctggcctggt atcagcagaa gcctggcaag gcccccaagc tgctgatctc tgaggccagc 3360 accctggaaa gaggcgtgcc cagcagattt tccggcagcg gctctggcac cgacttcacc 3420 ctgacaatca gcagcctgca gcccgaggac gtggccacct actactgcca gaagtacaac 3480 agcgtgcccc tgaccttcgg ccctggcacc aaggtggaaa tcaagccgcg ggccaacttt 3540 gtatacaaaa gtggcccgcg gacaacaacc cctgccccca gacctcctac cccagcccct 3600 acaattgcca gccagcctct gagcctgagg cccgaggctt gtagacctgc tgctggcgga 3660 gccgtgcaca ccagaggact ggatttcgcc tgcgacatct acatctgggc ccctctggcc 3720 ggcacatgtg gcgtgctgct gctgagcctc gtgatcaccc tgtactgcgg ctccaccagc 3780 ggctccggca agcccggctc tggcgagggc tccaccagcg gcgactacaa ggacgacgat 3840 gacaagtaat aggatatcgg ttcagctttc ttgtacaaag ttgggattcg agttaattaa 3900 gttaacgaat tccccccctc tccctccccc ccccctaacg ttactggccg aagccgcttg 3960 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc 4020 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc 4080 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa 4140 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct 4200 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca 4260 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca 4320 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat 4380 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc 4440 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc 4500 atgggaggcg gaagcggcgg aggctcccct cgaggcacca tggtgagcaa gggcgaggag 4560 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 4620 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 4680 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 4740 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 4800 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 4860 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 4920 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 4980 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 5040 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 5100 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 5160 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 5220 gccgggatca ctctcggcat ggacgagctg tacaagtaac gcgtcccggg tctagagcta 5280 gcggtaccat gcattacgta gtcgacgact taattaagct agcctagtgc catttgttca 5340 gtggttcgta gggctttccc ccactgtttg gctttcagtt atatggatga tgtggtattg 5400 ggggccaagt ctgtacagca tcttgagtcc ctttttaccg ctgttaccaa ttttcttttg 5460 tctttgggta tacatttaaa ccctaacaaa acaaagagat ggggttactc tctaaatttt 5520 atgggttatg tcattggatg ttatgggtcc ttgccacaag aacacatcat acaaaaaatc 5580 aaagaatgtt ttagaaaact tcctattaac aggcctattg attggaaagt atgtcaacga 5640 attgtgggtc ttttgggttt tgctgcccct tttacacaat gtggttatcc tgcgttgatg 5700 cctttgtatg catgtattca atctaagcag gctttcactt tctcgccaac ttacaaggcc 5760 tttctgtgta aacaatacct gaacctttac cccgttgccc ggcaacggcc aggtctgtgc 5820 caagtgtttg ctgacgcaac ccccactggc tggggcttgg tcatgggcca tcagcgcatg 5880 cgtggaacct tttcggctcc tctgccgatc catactgcgg aactcctagc cgcttgtttt 5940 gctcgcagca ggtctggagc aaacattatc gggactgata actctgttgt cctatcccgc 6000 aaatatacat cgtttccatg gctgctaggc tgtgctgcca actggatcct gcgcgggacg 6060 tcctttgttt acgtcccgtc ggcgctgaat cctgcggacg acccttctcg gggtcgcttg 6120 ggactctctc gtccccttct ccgtctgccg ttccgaccga ccacggggcg cacctctctt 6180 tacgcggact ccccgtctgt gccttctcat ctgccggacc gtgtgcactt cgcttcacct 6240 ctgcacgtcg catggagacc accgtgaacg cccaccaaat attgcccaag gtcttacata 6300 agaggactct tggactctca gcaatgtcaa cgaccgacct tgaggcatac ttcaaagact 6360 gtttgtttaa agactgggag gagttggggg aggagattag gttaaaggtc tttgtactag 6420 gaggctgtag gcataaattg gtctgcgcac cagcaccatg gcgcaatcac tagagcgggg 6480 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa 6540 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta 6600 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 6660 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 6720 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 6780 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat 6840 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 6900 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 6960 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc 7020 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 7080 ttatgcagag gccgaggccg gatcccttga gtggctttca tcctggagca gactttgcag 7140 tctgtggact gcaacacaac attgccttta tgtgtaactc ttggctgaag ctcttacacc 7200 aatgctgggg gacatgtacc tcccaggggc ccaggaagac tacgggaggc tacaccaacg 7260 tcaatcagag gggcctgtgt agctaccgat aagcggaccc tcaagagggc attagcaata 7320 gtgtttataa ggcccccttg ttaattcttg aagacgaaag ggcctcgtga tacgcctatt 7380 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 7440 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 7500 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 7560 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 7620 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 7680 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 7740 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 7800 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 7860 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 7920 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 7980 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 8040 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc 8100 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 8160 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 8220 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 8280 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 8340 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 8400 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 8460 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 8520 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 8580 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 8640 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 8700 cttcaggaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 8760 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 8820 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 8880 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 8940 gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga 9000 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 9060 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 9120 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 9180 caacgcggcc tttttacggt tcctggcctt ttgctggcct ttttgaagct gtccctgatg 9240 gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga 9300 agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcg 9344 SEQ ID NO: 39 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg   60 (pLenti-C-Myc- atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt  120 DDK OX40L) gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc  180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca  240 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata  300 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga  360 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt  420 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt  480 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca  540 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt  600 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt  660 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca  720 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg  780 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc  840 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta  900 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact  960 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1020 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1080 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1140 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1200 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 1260 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 1320 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1380 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1440 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1500 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1560 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1620 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1680 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1740 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1800 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1860 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1920 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1980 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2040 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2100 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2160 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580 cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3000 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240 tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt 3300 accgaggaga tctgccgccg cgatcgccat ggaaagggtc caacccctgg aagagaatgt 3360 gggaaatgca gccaggccaa gattcgagag gaacaagcta ttgctggtgg cctctgtaat 3420 tcagggactg gggctgctcc tgtgcttcac ctacatctgc ctgcacttct ctgctcttca 3480 ggtatcacat cggtatcctc gaattcaaag tatcaaagta caatttaccg aatataagaa 3540 ggagaaaggt ttcatcctca cttcccaaaa ggaggatgaa atcatgaagg tgcagaacaa 3600 ctcagtcatc atcaactgtg atgggtttta tctcatctcc ctgaagggct acttctccca 3660 ggaagtcaac attagccttc attaccagaa ggatgaggag cccctcttcc aactgaagaa 3720 ggtcaggtct gtcaactcct tgatggtggc ctctctgact tacaaagaca aagtctactt 3780 gaatgtgacc actgacaata cctccctgga tgacttccat gtgaatggcg gagaactgat 3840 tcttatccat caaaatcctg gtgaattctg tgtccttacg cgtacgcggc cgctcgagca 3900 gaaactcatc tcagaagagg atctggcagc aaatgatatc ctggattaca aggatgacga 3960 cgataaggtt taaacggccg gccgcggtct gtacaagtag gattcgtcga gggacctaat 4020 aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg gttccactag 4080 gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga 4140 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 4200 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 4260 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 4320 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 4380 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 4440 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 4500 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 4560 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 4620 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 4680 tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc 4740 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga 4800 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa 4860 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 4920 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc 4980 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg 5040 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac 5100 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga 5160 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg 5220 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 5280 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 5340 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 5400 tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5460 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 5520 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 5580 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5640 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5700 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5760 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5820 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5880 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5940 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 6000 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 6060 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 6120 tttggtcatg attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc 6180 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc 6240 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg 6300 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg 6360 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc 6420 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc 6480 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat 6540 atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg 6600 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa 6660 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat 6720 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt 6780 tttttctcca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6840 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cccgcgcaca 6900 tttccccgaa aagtgccacc tgac 6924

In the preparations of engineered EM-3 aAPCs (also referred to herein as aEM3 aAPCs) used for the experiments described herein, expression of CD86 and 4-1BBL was confirmed using flow cytometry (Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA), with results shown in FIG. 37. aEM3 aAPCs were γ-irradiated at 100 Gy and frozen.

aEM-3 cells previously transduced to express CD86, antibody against IgG Fc region, and 4-1BBL (or optionally without 4-1BBL), as described above, were genetically engineered with a co-stimulatory human OX-40L using a similar lentiviral transduction approach. To generate lentivirus containing human OX-40L, pLenti-C-Myc-DDK OX40L (PS100064, Origene, SEQ ID NO:39, FIG. 90) vector together with the VSV-G envelope plasmid (pCIGO-VSV.G) were co-transfected into a Phoenix-GP (ATCC CRL-3215) cell line using PolyJet (Signagen Laboratories, Rockville, Md., USA). The supernatants were harvested 60 hours later and concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane. aEM-3 cells were then infected with concentrated lentivirus and further expanded for five days. The cells were stained with PE-conjugated anti-human OX40L, Brilliant Violet 421-conjugated anti-human CD137L (if 4-1BBL is included in the prior aEM-3 cells), and PE/Cy7 conjugated anti-human CD86 and sorted based on the expression of GFP, OX40L, CD137L (when included), and CD86 using a S3e Cell Sorter (Bio-Rad, Inc., Hercules, Calif., USA). The purity of sorted cells was further validated using flow cytometry. The enriched cells were checked for purity by flow cytometry.

Example 6—Expansion of Tumor Infiltrating Lymphocytes Using EM-3 Artificial Antigen Presenting Cells

Experiments were performed to test the ability of EM-3 aAPCs (aEM3) to expand TILs. TIL were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 ratio plus OKT-3 (30 mg/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14. The results are plotted for two batches of TILs in FIG. 38 and FIG. 39. In addition, TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL) with or without OKT-3 (30 mg/mL). The results are plotted in FIG. 40, where the bar graph shows cell numbers determined on Day 11.

FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs (aEM3) at different TIL:aAPC ratios. The results show that aEM3 aAPCs perform comparably to and in some cases better than PBMCs, particularly at ratios of 1:200 at longer culture times (14 days).

FIG. 42 illustrates the low variability in cell counts from TIL expansions with EM-3 aAPCs (aEM3) in comparison to PBMC feeders. TILs (2×10⁴) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14. FIG. 43 compares the results of TIL expansions with EM-3 aAPCs and MOLM-14 aAPCs, to illustrate variability in cell counts for both aEM3 and aMOLM14 in comparison to TILs (2×10⁴) were co-cultured with five different PBMC feeder lots or aMOLM14 (in triplicate) or aEM3 (also in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. Viable cells were counted on day 14, and the graph shows viable cell numbers (mean) with 95% confidence interval. The aEM3 and aMOLM14 results indicate that much greater consistency can be obtained with both aAPCs compared to the PBMC feeder approach preferred in the prior art.

TILs expanded against aEM3 or PBMC feeders were used for flow cytometry analysis using 4 different panels (differentiation panels 1 and 2, T cell activation panels 1 and 2). Briefly, TILs were first stained with L/D Aqua to determine viability. Next, cells were surface stained with TCR α/β PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28 PE, CD27 APC-Cy7, and CD57-PerCP-Cy5.5 for differentiation panel 1; CD45RA PE-Cy7, CD8a PerCP/Cy5, CCR7 PE, CD4 FITC, CD3 APC-Cy7, CD38 APC, and HLA-DR PB, for differentiation panel 2; CD137 PE-Cy7, CD8a PerCP-Cy5.5, Lag3 PE, CD4 FITC, CD3 APC-Cy7, PD1 APC, and Tim-3 BV421 for T cell activation panel 1; or CD69 PE-Cy7, CD8a PerCP/Cy5.5, TIGIT PE, CD4 FITC, CD3 APC-Cy7, KLRG1 ALEXA 647, and CD154 BV421 for T cell activation panel 2. Phenotype analysis was done by gating 10,000 to 100,000 cells according to FSC/SSC using the Canto II flow cytometer. Data was analyzed using Cytobank software (Cytobank, Inc., Santa Clara, Calif., USA) to create sunburst diagrams and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) plots. Gates were set based on fluorescence minus one (FMO) controls. SPADE plots were generated with the group of cells, characterized in a form of related nodes based on the expression level of surface markers. CD4⁺ and CD8⁺ TIL subsets were determined based on CD3⁻ gating, and trees were generated. Sunburst visualizations are shown in FIG. 44 and FIG. 45. FIG. 44 shows that TILs expanded against aEM3 aAPCs maintained the CD8⁺ phenotype when compared to the same TILs expanded against PBMC feeders. FIG. 45 shows the results of a second batch of TILs from a different patient expanded against aEM3 aAPCs, where a clear increase of CD8⁺ cells (65.6%) is seen in comparison to the results from expansion using PBMC feeders (25%).

The CD4 and CD8 SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3⁺ cells is shown in FIG. 46 and FIG. 47. The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT. Without being bound by theory, the results show that TILs expanded with aEM3 aAPCs had undergone activation, but there was no difference in MFI between the aEM3 aAPCs and PBMC feeders, indicating that the aEM3 aAPCs effectively replicate the phenotypic results obtained with PBMC feeders.

Spare respiratory capacity (SRC) and glycolytic reserve were also evaluated for TILs expanded with aEM3 aAPCs in comparison to PBMC feeders, with results shown in FIG. 48 and FIG. 49. The Seahorse XF Cell Mito Stress Test measures mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells, using modulators of respiration that target components of the electron transport chain in the mitochondria. The test compounds (oligomycin, FCCP, and a mix of rotenone and antimycin A, described below) are serially injected to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using these parameters and basal respiration. Each modulator targets a specific component of the electron transport chain. Oligomycin inhibits ATP synthase (complex V) and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production. Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is uninhibited and oxygen is maximally consumed by complex IV. The FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity (SRC) is a measure of the ability of the cell to respond to increased energy demand. The third injection is a mix of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria.

FIG. 50 illustrates a mitochondrial stain of Live TILs expanded against PBMC feeders or aEM3 aAPCs. MitoTracker dye stains mitochondria in live cells and its accumulation is dependent upon membrane potential. TILs expanded against PBMC feeders or aEM3 were stained L/D Aqua followed by MitoTracker red dye. The data show MitoTracker positive (MFI) cells gated on live population,

Example 7—Comparison of Engineered MOLM-14 (aMOLM14) and EM-3 (aEM3) aAPCs

TILs expanded with PBMC feeders and aMOLM14 and aEM3 aAPCs, as described in the previous examples, were assessed for functional activity using the BRLA for cytotoxic potency. The P815 BRLA is described in detail in Example 9. The results are shown in FIG. 51 and FIG. 52, and show that TILs expanded with aAPCs have similar functional properties (and expected clinical efficacy) to those expanded with PBMC feeders.

IFN-γ release and Granzyme B release from TILs expanded with PBMC feeders and aMOLM14 and aEM3 aAPCs as described above was also assessed following overnight stimulation with microbeads coated with anti-CD3/CD28/4-1BB. The IFN-γ release results are shown in FIG. 53 and FIG. 54, and the Granzyme B release results are shown in FIG. 55 and FIG. 56. Significant and surprising increases in IFN-γ release and Granzyme B release were observed for TILs expanded with aEM3 aAPCs relative to those expanded with PBMC feeders, but not for TILs expanded by aMOLM14 aAPCs. Without being bound by theory, this suggests that TILs cultured with aEM3 aAPCs may be more active in vivo as a cancer therapy. Most other differences observed were not statistically significant.

The results of TIL expansions with aEM3 and aMOLM14 aAPCs are summarized in Table 9.

TABLE 9 Summary of TIL expansion results with aAPCs. Fold Expansion Relative CD8 (%) CD4 (%) Relative Relative aAPC TIL# PBMC aAPC expansion PBMC aAPC PBMC aAPC CD8 CD4 aMOLM14 M1032-T2 2112 1936 0.92 53 65 44 27 1.226 0.614 M1033-T6 1761 1598 0.91 50 57 36 40 1.140 1.111 M1021T-5 2053 2024 0.99 91 82  8 17 0.901 2.125 M1030T-4 860  853 0.99 46 78 51 12 1.696 0.235 M1045  858*  758* 0.88 — — — — — — M1021T-1 1866 1620 0.87 — — — — — — M1032T-1 2423 2049 0.85 — — — — — — M1042 1278 1704 1.33  8  8 88 89 0.919 1.015 M1043 1601 1587 0.99 90 87  5  5 0.968 0.947 aEM3 M1054 2058 1647 0.80 98 96  2  2 0.981 1.400 M1055  729 1533 2.10 25 66 70 31 2.694 0.441 M1021T-1 2985 2805 0.94 87 75 10 20 0.862 2.000 M1045 1336 1047 0.78 — — — — — —

Example 8—Preparation of Master Cell Banks for aEM3 and aMOLM14 aAPCs

aEM3 and aMOLM14 aAPCs may be grown in the following media compositions to produce master cell banks, which may be further grown in this media for supply of aAPCs: 500 mL of Dulbecco's Modified Eagle Medium DMEM/F12 (Sigma-Aldrich, St. Louis, Miss., USA), 50 mL fetal bovine serum (FBS) Heat Inactivated (HI) (Hyclone); 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) (Life Technologies); 1× Primocin (Invivogen); 1× Plasmocin (Invivogen), and 1× 2-mercaptoethanol (Life Technologies).

The aAPCs described herein, including aEM3 and aMOLM14 aAPCs, may also be grown from a master cell bank using any suitable method known in the art for the growth of cells. In an embodiment, aAPCs are thawed and are then expanded in a medium of 80-90% RPMI 1640+10-20% h.i. FBS (fetal bovine serum) by splitting saturated culture 1:2 to 1:3 every 2-3 days, seeding out at about 0.5-1×10⁶ cells/mL in 24-well plates, and maintaining at about 0.5-1.5×10⁶ cells/mL, with incubation at 37° C. and 5% CO₂.

Further steps that may be employed to use the aAPCs of certain embodiments of the present invention in the production of human therapies are known in the art and include cell line characterization (HLA high resolution typing); cytokine release testing; testing of human serum to replace FBS to grow aAPC; testing freezing media to freeze aAPCs; master cell banking (including raw material testing and stability testing); standardization of irradiation (including irradiation dose (1000, 3000, 5000, 10000, 15000 rad), fresh versus frozen aAPCs, and with/without TILs); stability of aAPC; development of a panel to evaluate the contamination of aAPCs; development of molecular biology assays (qPCR, DNA sequencing); testing of TIL expansions from different tumor types, including melanoma, cervical, and head and neck cancer (using a G-Rex 5M); potency, purity, and identity testing; mycoplasma and sterility assays; microbiological testing (USP/EP sterility, bioburden and endotoxin assays); and adventitious viral agent testing.

Example 9—Methods of Expanding TILs and Treating Cancer with Expanded TILs

TILs may be expanded using the aAPCs of certain embodiments of the present invention, such as aEM3 and aMOLM14 aAPCs, using any of the expansion methods described herein. For example, a method for expanding TILs is depicted in FIG. 57. The expansion of TILs using aAPCs may be further combined with any method of treating cancer in a patient described herein. A method for expanding TILs and treating a patient with expanded TILs, wherein the expansion makes use of aAPCs (including aEM3 and aMOLM14 aAPCs), is shown in FIG. 58.

Example 10—P815 Bioluminescent Redirected Lysis Assay

In this example, the development of a surrogate target cell line to evaluate the lytic potential of TILs in a Bioluminescent Redirected Lysis Assay (BRLA) is described. The BRLA enables assessment of T cell mediated killing in the absence of autologous tumor cells. Cytolytic activity can be assessed with and without engaging the T cell receptor in one to four hours, assessing T cell killing engaging the T cell receptor and without so-called lymphokine activated killer activities (LAK).

Mouse mastocytoma P815 cells expressing the endogenous CD16 Fc receptor can bind anti-CD3ε (OKT-3), providing a potent TCR activation signal as a target cell line. The P815 Clone G6 was transduced with a lentiviral vector based on eGFP and firefly luciferase, sorted and cloned using the BD FACSAria II. Clone G6 was selected based on eGFP intensity analyzed using an Intellicyt iQue Screener. Target cells and TILs of interest were co-cultured +/− OKT-3 to assess TCR activation (specific killing) or non-specific (lymphokine activated killing, LAK) respectively. Following 4 hours of incubation, firefly luciferin ((4S)-2-(6-hydroxy-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, commercially available from multiple sources) was added to the wells and incubated for 5 minutes. Bioluminescence intensity was read using a luminometer. Percent cytotoxicity and survival were calculated using the following formula: % Survival=(experimental survival−minimum)/(maximum signal−minimum signal)×100; % Cytotoxicity=100−(% Survival). Interferon gamma release in the media supernatant of co-cultured TILs was analyzed by ELISA, and LAMP1 (CD107a, clone eBioH4A3) expression on TILs was analyzed on a flow cytometer to evaluate the cytotoxic potency of TILs.

Results are shown in FIG. 59 to FIG. 75. FIG. 59 illustrates percent toxicity of TIL batch M1033T-1 co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios by BRLA. FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) data showing the amount of IFN-γ released against different ratios of effector to target cells. FIG. 61 illustrates LAMP1 (%) expressed by TIL batch M1033T-1 when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4 hours and 24 hours co-culture.

The results were confirmed using a second TIL batch as shown in FIG. 62, which illustrates BRLA for TIL batch M1030. The cytotoxicity (measured as LU₅₀/1×10⁶ TIL) by BRLA is 26±16. FIG. 63 illustrates the results of a standard chromium release assay for TIL batch M1030. The cytotoxicity (measured as LU₅₀/1×10⁶ TIL) by chromium release assay is 22.

Results were further confirmed using a third TIL batch. FIG. 64 illustrates BRLA results for TIL batch M1053, showing lytic units of the TILs by BRLA as 70±17. FIG. 65 illustrates the results of a standard chromium release assay for TIL batch M1053, showing lytic unit of the TILs by chromium assay as 14±5. Comparison of two assay results shows the comparable performance of the BRLA result to the chromium release assay result.

FIG. 66 illustrates the linear relationship between IFN-γ release and cytotoxic potential of TILs. FIG. 67 illustrates ELISpot results for IFN-γ. FIG. 68 illustrates enzymatic IFN-γ release for TIL batch M1053. FIG. 69 illustrates enzymatic IFN-γ release for TIL batch M1030. FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053T and M1030T. FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053. FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030. FIG. 73 illustrates ELISpot data showing TNF-α release by M1053T and M1030T. FIG. 74 illustrates enzymatic TNF-α release for TIL batch M1053. FIG. 75 illustrates enzymatic TNF-α release for TIL batch M1030. The data in FIG. 66 to FIG. 76 confirms the potency of these batches of TILs as also shown by the BRLA.

In conclusion, the BRLA requires no radionuclides and is as efficient and sensitive as traditional cytotoxicity assays. Flow cytometric assessment of Lamp1 expression on TILs at individual time points demonstrates degranulation of cytotoxic T cells relative to the potency shown by BRLA. The BRLA demonstrates similar to better potency than standard chromium release assay. BRLA also enables evaluation of the potency of TIL lytic activity. Comparison of BRLA with chromium release assay shows the efficiency and reliability of BRLA. BRLA has a linear relationship with IFNγ release by TILs. Release assay of IFN-γ, TNFα and Granzyme B by ELISpot is consistent with the cytotoxic efficiency of the TILs evaluated by BRLA.

Example 11—Process for Weaning EM3 Cells from FBS to hAB Serum

In order to avoid reactivity, some cell lines may need to be weaned from one medium to another. Here, EM3 cells are weaned from FBS to hAB serum to avoid reactivity. As shown in FIG. 76, aEM3 cells were successfully weaned off of FBS to hAB serum.

Example 12—Freezing Media Formulation Optimization

To cryobank EM3 cells cultured as described herein, methods were freezing media formulation were optimized. As shown in FIG. 77, three freezing media were used and their effect on cell numbers were counted. The cell media utilized included CryStor 10 (Biolife Solutions (CS10)) (A), hAB [90%] and DMSO [10%] (B), and hAB [20%] with DMSO [10%] and cDMEM2 [70%] (C). FIG. 77 demonstrates that the formulation of human AB serum (90%) and DMSO (10%) provided for unexpectedly increased EM3 cell numbers after 3 days of recovery.

Example 13—Growth of aEM3 Cells in GREX Flasks

aEM3 cells were cultured in gas permeable cell culture flasks (i.e., GREX flasks (Wilson Wolf Manufacturing)) and the effect on cell doubling time was observed over an 8 day time course. As shown in FIG. 78, the GREX flasks provided for rapid growth of aEM3 cells.

Example 14—Flow Panel Analysis to Determine aEM3 Cell Purity

To determine the purity of cells cultured according to the processes described herein, a flow panel analysis was used to determine the purity of aEM3 aAPCs. The results of such analysis are described in FIGS. 79 and 80. As shown in FIG. 80, before sorting aEM3 cell populations were 53.5% and 43.2% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively. Postsorting, cell populations was improved to 96.8% and 96.3% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively (FIG. 80).

Example 15—aEM3 Feeder Cells as an Alternative to PBMC Feeders

As described herein, aEM3 cells may be used as an alternative for PBMC feeders, resulting in unexpectedly different properties for both TIL expansion process and the resulting TILs. To compare differences in cytokine expression, PBMCs and aEM3 cells were stimulated by treatment with OKT-3. As shown in FIG. 81, aEM3 cells displayed a comparatively different cytokine expression profile as compared to PBMCs. Surprisingly, the aEM3 cells of the present invention provide efficacious TILs (as shown herein) without reproducing the same cytokine secretion properties of TILs expanded with conventional PBMCs.

Example 16—Comparison Between Complete Media and Serum Free Media TIL Expansion

In order to optimize the TIL expansion protocols, several TIL expansion experiments were performed as described herein, but with serum free media rather than complete media (CM1).

In one experiment, tissue fragments were cultured in a single well with CM1 or various serum free media with 300 IU/mL of IL-2. Cells were then counted on Day 11 before initiating REP. The various serum free media used included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), and Xvivo-20 (Lonza). As shown in FIG. 82, TIL expansion (PreREP) with CTS provided increased cell numbers as compared to CM1.

Additionally, tissue fragments were cultured with CM1 or various serum free media with 6000 IU/mL of IL-2 until Day 11. REP was then initiated on Day 11 using PBMC feeders, OKT-3, and IL-2, and culture was split on Day 16. Cultures were then terminated at the end of Day 22. The various serum free media used included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), and Xvivo-20 (Lonza). As shown in FIG. 83 and FIG. 84, when counting cells at Days 11 and Day 22, respectively, TIL expansion (PreREP) with Prime CDM provided increased cell numbers as compared to CM1.

Example 17—Growth of aAPCs in Serum Free Media as Compared to Serum-Based Media

In order to optimize aAPC growth and maintenance protocols in the absence of serum, aEM3 cells were cultured using various serum free media.

aEM3 cells were cultured in 24 well plates at 1×10⁶ cells per well for 3 days using general cell culture protocols as described herein, with the exception that that one group of cells were provided with serum-based media (cDMEM (10% hSerum) and the other groups of cells were provided with serum free media. The serum free media utilized for the study included CTS OpTmizer (ThermoFisher), Xvivo 20 (Lonza), Prime-TCDM (Irvine), and XFSM (MesenCult) media. Cells were then counted on Day 3.

As shown in FIG. 85, CTS OpTmizer and Prime-TCDM serum free media provided cell growth that was comparable to serum-based media (i.e., cDMEM (10% hSerum). Therefore, serum free media is an effective alternative for growing and maintaining aAPCs as compared to serum-based media.

Example 18—Propagation, Maintenance, and Cryopreservation of aAPCs

In this example, procedures are provided for the preparation and preservation of aAPCs. Specifically, aEM3 cells from a cell line designated TIL-Rs3 were propagated and cryopreserved.

Thawing and recovery of aEM3 cells may be accomplished using the following non-limiting procedure. Cyropreserved aEM3 cells are warmed slowly in pre-warmed media (37° C.) that is prepared from CTS OpTmizer Basal Media (Thermo Fisher), CTS OpTmizer Cell Supplement (Thermo Fisher), Gentamicin (Lonza), and Glutamax (Life Technologies). The suspended cells are then centrifuged at 1500 rpm for 5 minutes at 4° C. The resulting supernatant is discarded and the remaining aEM3 cells are resuspended in the foregoing media and plated (5×10⁶ cells/10 mL per well of a 6 well plate).

Propagation of aEM3 cells may be accomplished using the following non-limiting procedure. Aliquots of the foregoing media are prepared in gas permeable cell culture flasks (i.e., GREX 10 flasks (Wilson Wolf Manufacturing)). The plated aEM3 cells are washed by centrifugation (i.e., 1500 rpm for 5 minutes at 4° C.), resuspended in media, and added to the GREX flasks at cell density of 1-2×10⁶ cells/mL. The aEM3 cell suspension was diluted with 30 mL of media and the GREX flasks were then incubated for 3-4 days at 37° C. under CO₂. After 3-4 days, the GREX flasks were removed from the incubator and placed in a biological safety cabinet (BSC). The cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the increased number of aEM3 cells, which may be resuspended at a cell density of 10-20×10⁶ cells per GREX 10 flask.

An alternative cryopreservation of aEM3 cells may be accomplished using the following non-limiting procedure. The foregoing GREX 10 flasks containing the aEM3 cells are removed from the incubator and placed in a BSC. The cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the increased number of aEM3 cells, which is resuspended in a volume of CryStor 10 (Biolife Solutions) to provide a concentration of 10-100×10⁶ cells/vial in cryovials. The aEM3 cell suspensions may be placed in a freezing container and transferred to a −80° C. freezer.

Example 19—Demonstration of Rapid Recovery of aEM3 Cells Following Cryopreservation

aEM3 cells from the TIL-R3 cell line (1-2×10⁶ cells) were cryopreserved according to the procedure set forth in Example 18 using CS-10 cryopreservation media. Vials of such cells were then thawed and the cells were counted. Cell counts were taken pre-freeze, post-thaw, and 3 days after thaw (i.e., Post-Thaw Recovery). As shown in FIG. 86 and FIG. 87, the total live cell counts recovered rapidly post thaw in two separate experiments.

TIL-R3 cells (1×10⁶ cells) were thawed (Day 3 post-thaw) and plated at a density of 0.5×10⁶/cm² in each well of a 24 well plate. On day 0 and 3, viable cells were counted and recorded. On the first passage (Day 6), cells were split at the density of 2×10⁶ cells/cm² or 0.5×10⁶ cells/cm². At the end of the first passage, a cell count was performed. The resulting cell counts are shown in FIG. 88, which demonstrate both a recovery phase post-thaw and a growth phase.

Furthermore, TIL-R3 cells (20×10⁶ cells) were cultured at a density of 2×10⁶/cm² in GREX 10 flasks according to the procedure described in Example 18. On days 4 and 8, live cells were counted and recorded. The resulting cell counts are shown in FIG. 89, which demonstrates a growth phase for the cells following cryopreservation that reaches a plateau between days 4 and 8 when the cells reached a density of 13.9×10⁶ cells/cm².

Example 20—CD8 Skewness, Expansion Performance, and CD3 Contamination of TILs Cultured with aEM3 aAPCs

Fifteen different PreREP TIL lines (0.4×10⁵ cells) were co-cultured with either aEM3 aAPCs (as described herein) or PBMC feeders (10×10⁶), OKT3 (30 ng/mL) and IL-2 (3000 IU/mL) and cultures were split on Day 5 using 6 well Grex plates. Cultures were sampled at day 11 and analyzed by flow cytometry. The relative ratio of CD8⁻ cells was calculated by the formula (CD8% aEM3)/(CD8% PBMC). The results shown in FIG. 91 indicate that TILs cultured with aEM3 cells surprisingly promote CD8⁺ skewing and and an improved TIL product. Additional results of these experiments are shown in FIG. 92, FIG. 93, and FIG. 94, where the results shown that TILs cultured with aEM3 aAPCs displayed comparable expansion and less non-CD3+ cell contamination in comparison to TILs cultured with PBMC feeders.

Example 21—Telomere Length Measurement

Genomic DNA was isolated from pre-REP or post-REP (magnetic bead sorted for CD3⁺) TILs for a qPCR (quantitative polymerase chain reaction) assay to measure telomere length. The real time qPCR method is described in Cawthon, Nucleic Acids Res. 2002, 30(10), e47; and Yang, et al., Leukemia, 2013, 27, 897-906. Briefly, the telomere repeat copy number to single gene copy number (T/S) ratio was determined using an PCR thermal cycler (Bio-Rad Laboratories, Inc.) in a 96-well format. Ten ng of genomic DNA was used for either the telomere or hemoglobin (hgb) PCR reaction and the primers used were as follows:

-   Tel-1b primer (CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT)     (SEQ ID NO:40); -   Tel-2b primer (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT)     (SEQ ID NO:41); -   hgb1 primer (GCT TCT GAC ACA ACT GTG TTC ACT AGC) (SEQ ID NO:42);     and -   hgb2 primer (CAC CAA CTT CAT CCA CGT TCA CC) (SEQ ID NO:43).

All samples were analyzed by both the telomere and hemoglobin reactions, and the analysis was performed in triplicate on the same plate. In addition to the test samples, each 96-well plate contained a five-point standard curve from 0.08 ng to 250 ng using genomic DNA isolated from the 1301 human T-cell leukemia cell line (available from Sigma and ATCC). The T/S ratio (−dCt) for each sample was calculated by subtracting the median hemoglobin threshold cycle (Ct) value from the median telomere Ct value. The relative T/S ratio (−ddCt) was determined by subtracting the T/S ratio of the 10.0 ng standard curve point from the T/S ratio of each unknown sample.

Results are shown in FIG. 95. Each data point shown is the median measurement of relative T/S ratio. The results indicate that TILs cultured with aEM3 maintain their telomere length. 

We claim:
 1. An isolated artificial antigen presenting cell (aAPC) comprising an EM-3 myeloid cell that endogenously expresses ICOS-L (inducible T-cell co-stimulator ligand), CD58, and one or more of HLA (human leukocyte antigen)-A, HLA-B, or HLA-C, wherein said aAPC is stably transduced with one or more viral vectors, wherein the one or more viral vectors comprise: (i) a nucleic acid encoding CD86; (ii) one or more nucleic acids encoding one or more costimulatory molecules selected from the group consisting of OX40L and 4-1BBL; and, (iii) a nucleic acid encoding SEQ ID NO:27; wherein the myeloid cell expresses on its surface a protein encoded by each of the nucleic acids of (i), (ii) and (iii).
 2. The aAPC of claim 1, wherein the aAPC can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the aAPC.
 3. The aAPC of claim 1, wherein the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 (Interleukin-2) at a concentration of about 3000 IU/mL and OKT-3 antibody at a concentration of about 30 ng/mL.
 4. The aAPC of claim 1, wherein the aAPC can stimulate and expand a T cell contacted with the aAPC.
 5. The aAPC of claim 1, wherein the CD86 protein comprises a sequence as set forth in SEQ ID NO:8, or a sequence comprising one or more conservative amino acid substitutions thereof.
 6. The aAPC of claim 1, wherein the nucleic acid encoding CD86 comprises SEQ ID NO:19.
 7. The aAPC of claim 1, wherein the one or more costimulatory molecules comprises a 4-1BBL protein.
 8. The aAPC of claim 7, wherein the 4-1BBL protein comprises a sequence as set forth in SEQ ID NO:9, or a sequence comprising one or more conservative amino acid substitutions thereof.
 9. The aAPC of claim 7, wherein the one or more nucleic acids encoding the 4-1BBL protein comprises SEQ ID NO:16.
 10. The aAPC of claim 1, wherein the one or more costimulatory molecules comprises an OX40L protein.
 11. The aAPC of claim 10, wherein the OX40L protein comprises a sequence as set forth in SEQ ID NO:10, or a sequence comprising one or more conservative amino acid substitutions thereof.
 12. An isolated artificial antigen presenting cell (aAPC) comprising an EM-3 cell that endogenously expresses ICOS-L (inducible T-cell co-stimulator ligand), CD58, and one or more of HLA-A, HLA-B, or HLA-C, wherein said aAPC is stably transduced with one or more viral vectors, wherein the one or more viral vectors comprise: (i) a nucleic acid encoding CD86 or more conservative amino acid substitutions thereof; (ii) one or more nucleic acids comprising a sequence encoding one or more amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:14; (iii) a nucleic acid encoding SEQ ID NO:27; wherein the EM-3 cell expresses on its surface a protein encoded by each of the nucleic acids of (i), (ii) and (iii). 